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Receptor and STAT11
* Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021;
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021; and
Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York, NY 10016
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Abstract |
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. Mice deficient in both TLR2 and TLR4 handle aerosol infection with viable Mtb as well as congenic controls. Viable Mtb also up-regulates inducible NO synthase, RANTES, IFN-inducible protein 10, and IRG1 in macrophages that lack mannose receptor, complement receptors 3 and 4, type A scavenger receptor, or CD40. These MyD88, TLR2/4-independent transcriptional responses require IFN-
R and STAT1, but not IFN-
. Conversely, those genes whose expression is MyD88 dependent do not depend on IFN-R or STAT1. Transcriptional induction of TNF is TLR2/4, MyD88, STAT1, and IFN-R independent, but TNF protein release requires the TLR2/4-MyD88 pathway. Thus, macrophages respond transcriptionally to viable Mtb through at least three pathways. TLR2 mediates the responses of a numerically minor set of genes that collectively do not appear to affect the course of infection in mice; regulation of TNF requires TLR2/4 for post-transcriptional control, but not for transcriptional induction; and many responding genes are regulated through an unknown, TLR2/4-independent pathway that may involve IFN-R and STAT1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and CD40L from NK and Th1 cells, macrophages can kill intracellular Mtb, in part through induction of inducible NO synthase (iNOS) (reviewed in Ref.1). Several different macrophage receptors mediate Mtb binding and uptake (2), and macrophages respond to Mtb by dramatically reprogramming their transcriptome (3). However, there is little evidence to establish a mechanistic connection between surface receptors and transcriptional remodeling when macrophages encounter viable, virulent Mtb.
Macrophage receptors that participate in the uptake of Mtb include complement receptors (CR3, CD11b/CD18; CR4, CD11c/CD18), mannose receptor (MR), and scavenger receptors. However, aside from matrix metalloprotease 9 induction, there is little evidence that these receptors mediate signaling events during the encounter of macrophage and live Mtb. Conversely, TLRs and CD40 have not been implicated in the uptake of Mtb but do transduce signals in macrophages in response to certain Mtb subcellular products. The TLR pathway can be implicated by disrupting individual TLRs or their adaptor proteins, such as MyD88, Toll-IL-1R (TIR) domain-containing adaptor protein (TIRAP), and TIR domain-containing adaptor inducing IFN- (TRIF). Engagement of TLRs can lead to the transcription of proinflammatory cytokine genes and costimulatory molecules. For example, the mouse macrophage-like cell line RAW-TT10 responded to heat-killed Mtb H37Rv, H37Ra, and mycobacterial cell wall fractions enriched in lipoarabinomannan, mycolylarabinogalactan-peptidoglycan complexes, or other lipids to produce TNF in a TLR2-dependent manner (4). Overexpression of TLR2 and TLR4 allowed Chinese hamster ovary cells and RAW264.7 macrophage-like cells to respond to live Mtb via both TLR2 and TLR4 (5). Mycobacterial cell wall glycolipid lipoarabinomannan, mannosylated phosphatidylinositol, and a 19-kDa Mtb lipoprotein served as TLR2 agonists on Chinese hamster ovary cells and RAW cells transfected with TLR2, whereas an undefined heat-labile moiety of Mtb acted via TLR4 (5). Activation of human macrophages via TLR2 in response to a 19-kDa Mtb lipoprotein led to NO-independent inhibition of Mtb replication (6). Very few such experiments, however, have used intact, viable, virulent Mtb and primary macrophages expressing only endogenous receptors at a physiologic level. Our own experiments indicated that MyD88 is dispensable for the majority of transcriptional responses of primary mouse macrophages to live, virulent Mtb (7). This implied either that TLRs are not the major receptors used by those cells for recognizing intact, live Mtb, or that TLR-dependent responses to Mtb mostly involve MyD88-independent signaling pathways. The latter explanation would be unprecedented for TLR2, for which MyD88 serves as an indispensable adaptor. Ambiguity concerning the roles of TLR2 and TLR4 in Mtb infection has been compounded by discordant results in mice that express a mutant form of TLR4 (the C3H/HeJ allele) or bear disrupted alleles for TLR2 (8, 9, 10, 11, 12).
To clarify the roles that TLR2 and TLR4 play in macrophage responses to viable Mtb, we assessed global gene expression in Mtb-infected TLR2-deficient (TLR2–/–) mouse macrophages by microarray, susceptibility of TLR2/TLR4-deficient mice to aerosol infection with Mtb, and gene expression in Mtb-infected bone marrow-derived macrophages (BMM) from mice deficient in TLR2, TLR4, both TLR2 and TLR4, TIRAP, or TRIF. These studies were also extended to macrophages deficient in MR, CD18 (and thus both CR3 and CR4), type A scavenger receptor (SR-A), and CD40. Finally, because MyD88-independent expression of MCP-5, iNOS, and IFN-inducible protein 10 (IP10) in primary mouse macrophages in response to TLR4 engagement depended on IFN- and STAT1 (13), we also examined the roles of IFN-, IFN-R, and STAT1. Results from our analyses suggest that neither TLR2 nor TLR4 is required for host defense against Mtb and that neither TLR2, TLR4, MyD88, TIRAP, nor TRIF is required for transcriptional induction of iNOS, immune-responsive gene 1 (IRG1), RANTES, and IP10 by live Mtb in primary BMM in vitro. In contrast, these TLR2/4, MyD88-independent, Mtb-responsive genes require IFN-R and STAT1 for their expression in Mtb-infected macrophages. The pathway by which macrophages sense Mtb and respond via type I IFN signaling does not involve MR, SR-A, CD18 (including CR3/4), or CD40 and remains to be defined. TLR2/4- and MyD88-dependent responses can be subdivided into those that require TLR2/4 and MyD88 for transcriptional induction and those that depend on this pathway for post-transcriptional control.
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Materials and Methods |
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Drs. K. Takeda and S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) provided fertilized MyD88–/+ ova derived from C57BL/6 mice and MyD88–/– mice that had been backcrossed to C57BL/6 mice for six generations. The mice to which the zygotes gave rise were intercrossed to generate MyD88–/– and wild-type (wt) control mice. TIRAP–/– mice were provided by Dr. R. Medzhitov (Yale University School of Medicine, New Haven, CT) (14). TRIF-mutant mice were provided by Dr. B. Beutler (The Scripps Institute, La Jolla CA) (15). TLR2–/– and TLR4–/– mice (16, 17) were also provided by Dr. S. Akira and were crossed to obtain TLR2/TLR4 double-deficient mice. Deficiency of both TLRs was confirmed by PCR from genomic DNA and lack of response of BMM to TLR2-activating synthetic triacylated peptide Pam3CSK4 and TLR4-activating LPS. STAT1–/– and IFN-R–/– mice were generated as previously described (18, 19). IFN-–/–, CD18–/–, and CD40–/– mice were purchased from The Jackson Laboratory. MR knockout mice and SR-A knockout mice were generated as previously described (20, 21). Controls were mice of the same genetic background. Mice were housed under specific pathogen-free conditions.
Activation and infection of primary mouse macrophages
For all in vitro studies except microarrays, macrophages were collected in two or three independent experiments from 8- to 10-wk-old gene-deficient mice and control mice on the same background (three mice per experiment). Bone marrow cells were flushed from femurs and differentiated into macrophages as previously described (3). This resulted in a highly homogeneous cell population, as judged by the expression of surface markers characteristic of macrophages (CD14, Mac-1, CD18, CD16/32, and F4/80) and as assessed by flow cytometry (not shown). Macrophages were infected with Mtb from early log phase cultures of a low passage clinical isolate (strain 1254; American Type Culture Collection 51910) at a multiplicity of infection (MOI) of 5 as described previously (3). Intracellular survival of Mtb and measurement of nitrite in the conditioned medium were previously reported (3). For microarrays, there were six independent experiments. The six experiments were independent in the sense that they involved collection of cells from different pools of mice (two mice of each genotype per pool) on different days, preparation of cDNA on different days, and hybridization to different arrays on different days.
Mouse infections
Mice were infected with logarithmic phase cultures of Mtb by aerosol using an inhalation exposure system (Glas-Col). Animals were exposed for 40 min to an aerosol produced by nebulizing 5 ml of a bacterial suspension in PBS at a concentration of 
2 x 107 bacilli/ml and 2 x 108 bacilli/ml. This resulted in inoculum sizes of 50–70 and 600–700 CFU/lung as determined by plating homogenized lungs onto enriched 7H11 plates 24 h after infection.
Quantification of viable Mtb in mouse organs
Mice were killed by inhalation of CO2 under noncrowded conditions. Lungs were aseptically removed and homogenized in 4 ml of PBS containing 0.05% Tween 80. On day 1 after infection, 1.6 ml of lung homogenate were plated on four 7H11 agar (Difco) plates supplemented with 10% oleic acid/albumin/dextrose catalase and 0.5% glycerol. Plates were incubated at 37°C, and CFU were enumerated 14–21 days later. Thereafter, at indicated time points, mice were killed, and their lungs and spleens were aseptically removed. Spleens and one lobe of the lung were homogenized in PBS containing 0.05% Tween 80, and serial dilutions were plated on enriched 7H11 plates for CFU. The other lobes were homogenized in 4 ml of TRIzol for RNA preparation.
Array hybridization
Twenty-four hours after infection, macrophage monolayers were lysed with TRIzol (Invitrogen Life Technologies), and total RNA was isolated. After treatment with DNase I (Ambion) and purification (RNeasy; Qiagen), RNA (2–3 µg) was reverse transcribed (SuperScript II; Invitrogen Life Technologies) with a T7-poly(T) primer, and cDNA was transcribed in the presence of biotinylated UTP and CTP (Enzo). Hybridization to GeneChip oligonucleotide arrays (Mu11KsubA, B) and scanning (Gene-Array scanner) followed Affymetrix protocols.
Data processing
Primary image analysis of the arrays was performed using GeneChip Microarray Analysis Suite version 5.0 (Affymetrix), and images were scaled to an average hybridization intensity (average difference) of 250. Data analysis was conducted using GeneSpring 6.2 software (Silicon Genetics). Normalization was applied using the distribution of all genes on each chip, i.e., all measurements on each chip were divided by the 50th percentile value of that chip. Next, each gene was compared with its control by dividing its intensity by the average intensity of that gene in the six control samples (untreated macrophages). Data from six independent replicate experiments were used to perform a Wilcoxon two-sample rank test for each gene. Data from a total of 24 arrays (two genotypes, wt and TLR2–/–; two conditions, untreated and Mtb infected; six replicates for each condition and genotype) were included in the analysis. Of the 11,000 genes represented on the Affymetrix arrays, 6640 genes were detected as present (detection p < 0.04) by the Affymetrix Microarray Suite software in at least four of six samples in one of the two conditions compared. These 6640 genes were included in additional analysis; the other genes were excluded because of their low, and thus unreliable, signal values. The regulation of each of these 6640 genes was tested in a Wilcoxon two-sample rank test in wt macrophages and TLR2–/– macrophages. A gene was considered regulated compared with its control if its regulation changed across six experiments with p 
0.05. The Wilcoxon two-sample rank test was chosen because it is a nonparametric test that compares two paired groups. It makes no rigid assumptions about the distribution of the tested populations (they do not have to follow a Gaussian distribution) and is resilient to outlier data. To identify TLR2-dependent and TLR2-independent genes, regulation factors (absolute signal intensities in response to a stimulus divided by signal intensities of untreated samples) were tested with a Wilcoxon two-sample rank test for each gene in wt vs TLR2–/– macrophages, with p 0.05 as the cut-off for statistical significance. Data mining was conducted with FileMakerPro 6. This database program allowed to organize the data and to query for data that meet several criteria. For example, TLR2-dependent genes were regulated in wt macrophages with p 0.05, but either were not regulated in TLR2–/– macrophages (p > 0.05) or were regulated with a fold change that was at least 2-fold reduced in the TLR2–/– macrophages (p 0.05 and fold regulation in wt/fold regulation in TLR2–/– 
2; see Results).
Quantitative real-time PCR (qRT-PCR)
RNA was prepared from Mtb-infected BMM, Mtb-infected mouse lungs, as well as uninfected control cells and lungs. Two hundred nanograms of RNA was transcribed into cDNA with gene-specific primers in 20 µl using 50 U of Moloney murine leukemia virus reverse transcriptase (PerkinElmer). cDNA was diluted to 100 µl. PCR was performed in a volume of 15 µl on the ABI PRISM 7900HT sequence detection system (PerkinElmer) as previously described (7). The sequences of primers and probes, except that for IFN-, were previously described (1). The sequences of primers and probes for IFN- were: forward primer, 5'-CTGGAGCAGCTGAATGGAAAG-3'; reverse primer, 5'-TCTCCGTCATCTCATAGGGA-3'; and probe, 5'FAM-TCAACCTCACCTACAGGGCGGACTTC-3' black whole quencher.
Quantification of TNF and IL-10 protein release by ELISA
BMM from wt, MyD88–/–, and TLR2/TLR4–/– mice were seeded in 24-well plates at 5 x 105 cells/well. Cells were infected with live Mtb at a MOI of 3–5 as previously described (3). Supernatants were collected 24 h after infection. Mouse TNF and IL-10 concentrations were measured by ELISA according to the manufacturer’s instructions (R&D Systems).
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Results |
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Many reports have concluded that macrophages respond to Mtb products via TLR2, yet viable Mtb regulates mouse macrophage gene expression in a manner largely independent of MyD88 (7), a molecule on which all TLR2-mediated responses are thought to depend. To solve this puzzle, we first examined gene expression in Mtb-infected TLR2–/– mouse BMM by microarray analysis of global gene expression. Gene expression in response to Mtb in the absence of TLR2 was compared with that of wt and MyD88–/– macrophages by Wilcoxon two-sample rank test (7). Of the 6640 genes included in the analysis, 286 genes were regulated in both wt and TLR2–/– macrophages, and their fold change in regulation was not >2-fold different between the two genotypes. These genes were classified as TLR2 independent (Fig. 1A and Supplemental Table IA4). Of these genes, 164 were also MyD88 independent (7). In contrast, there were only 71 genes that were regulated in wt macrophages, but either not regulated in TLR2–/– macrophages or regulated with a fold change that was at least 2-fold reduced in TLR2–/– macrophages. These were classified as TLR2 dependent (Fig. 1A and Supplemental Table IB). Most of these were also MyD88 dependent. Independent analyses of the microarray data using Welch’s approximate t test for two groups and Student’s two-sample t test identified similar numbers of TLR2-dependent and TLR2-independent genes. Thus, more than twice as many genes required neither TLR2 nor MyD88 for their regulation by Mtb than those that were MyD88 and TLR2 dependent.
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Leaving aside the role of MyD88, we focused on specific examples of immunologically important genes to confirm TLR2-independent signaling in response to viable Mtb. For this, we applied qRT-PCR to mRNA from wt and TLR2–/– macrophages. This set of experiments also included TLR4–/– and TLR2/TLR4–/– macrophages to evaluate possible redundancy between TLR2 and TLR4 (Fig. 1B). Indeed, the expression of iNOS, IP10, RANTES, and IRG1 was regulated by Mtb in the absence of either or both TLR2 and TLR4.
Expression of iNOS and IRG1 was also analyzed in the lungs of mice that were infected by aerosol with 50–70 CFU of virulent Mtb. Gene expressions in lungs of wt, TLR2–/–, and TLR2/TLR4–/– mice were similar (Fig. 2A). Moreover, wt and TLR2/TLR4–/– mice sustained similar bacterial loads in lungs (Fig. 2B) and spleens (not shown), and Mtb-infected mice of both genotypes died between 11 and 13 mo after infection, with similar mean times to death. In a second experiment, wt and TLR2/TLR4–/– mice were infected with 600 CFU. Despite the higher inoculum, there was no significant difference in gene expression (Fig. 2C) or bacterial loads in lungs (Fig. 2D) and spleens (not shown), and mice of both genotypes died between 7 and 11 mo after infection with similar mean times to death. In both experiments, the histopathologic reaction, including granuloma formation, was indistinguishable in infected lungs from wt and TRL2/TLR4–/– mice (not shown). The lack of impact of deficiency of TLR2 and TLR4 contrasted with the marked susceptibility of MyD88–/– mice to Mtb, as observed in two other studies (22, 23) and confirmed in this study (Fig. 2, B and D).
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MyD88-independent, TLR2/TLR4-independent gene regulation in response to Mtb is not mediated by TIRAP or TRIF
TIRAP associates with TLR4 and participates in TLR4-mediated gene expression in response to bacterial LPS (24). Studies with TIRAP-deficient mice suggested that TIRAP is involved in MyD88-dependent signaling downstream of TLR2 and TLR4 (14, 25). TRIF transmits TLR3-mediated signals, accounting for MyD88-independent responses elicited by dsRNA (26), and plays a role in MyD88-independent signaling via TLR4 (15, 27). The TRIF-mediated pathway triggers production of type I IFN, leading to the expression of IFN-inducible genes. However, as shown in Fig. 3, after infection with viable Mtb, the expression of the MyD88-independent genes iNOS, IP10, RANTES, and IRG1 was not significantly impaired in macrophages from TIRAP- or TRIF-deficient mice. The expression of iNOS was 3.6-fold reduced in Mtb-infected MyD88–/– macrophages compared with infected wt macrophages. However, the expression of iNOS in Mtb-infected MyD88–/– macrophages was still greater than that in uninfected macrophages by a factor of >10. Thus, induction of iNOS, IP10, RANTES, and IRG1 by Mtb is mediated in large part via a pathway that does not require TLR2, TLR4, MyD88, TIRAP, or TRIF.
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Blocking of MR, CR3/4, or SR-A almost completely abrogated binding of Mtb to human macrophages (28). MR participates in Mtb induced up-regulation of metalloproteinase 9 (29), but the roles of CR and SR-A in signal transduction or cell activation in response to Mtb have not been characterized. CD40 mediates Mtb 70-kDa heat shock protein-induced production of RANTES, MIP-1, and MIP-1 in human monocytes and production of IL-12 in murine dendritic cells (DC) (30, 31). We infected BMM from MR–/–, CD18–/–, CD40–/–, and SR-A–/– mice with viable Mtb and assessed the expression of iNOS, IP10, RANTES, and IRG1. Mtb regulated the expression of these genes to the same extent as in wt macrophages in the absence of any one of these receptors (Fig. 4; data not shown for CD18 and CD40). Thus, each of these receptors is dispensable under the conditions and for the responses studied.
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R and STAT1 are critical for expression of iNOS, IP10, RANTES, and IRG1 in Mtb-infected macrophages
Induction of iNOS, IP10, RANTES, and IRG1 mRNA expression in response to Mtb was detectable after 4 h, but not after 1 h, and could be blocked by cycloheximide (not shown). These observations suggested that protein synthesis was required for macrophages to transcribe these genes in response to Mtb. Because IFN- mediates some MyD88-independent gene regulation in response to TLR3 or TLR4 ligands (15, 26, 27), we asked whether type I IFN might also contribute to Mtb-regulated gene expression. In fact, Mtb induced IFN in macrophages from wt, TLR2/4–/–, MyD88–/–, TIRAP–/–, and TRIF–/– mice (Fig. 5). Moreover, the expression of MyD88-independent genes in IFN-R–/– and STAT1–/– macrophages after infection with Mtb was markedly reduced compared with that in wt macrophages, whereas gene expression in IFN-–/– macrophages was not affected (Fig. 6A). STAT1 was also critical for controlling the growth of intracellular Mtb in macrophages (Fig. 6B). These data suggest that Mtb induces a signal transduction pathway in macrophages that depends on IFN-R.
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Mtb-stimulated production of TNF was significantly reduced in primary mouse macrophages that lacked TLR2 (11). We investigated whether the induction of TNF mRNA also required TLR2 or TLR4. Fig. 7A shows that transcription of TNF was up-regulated by Mtb even in the absence of MyD88 and TLR2/TLR4. In contrast, protein production or release was significantly impaired in MyD88–/– and TLR2/TLR4–/– macrophages (Fig. 7B). This demonstrates that TLRs can be differentially involved in mediating gene induction at the transcriptional level and in post-transcriptional control. In contrast to the TLR2/4-independent genes iNOS, IP10, RANTES, and IRG1, TNF mRNA expression in Mtb-infected macrophages did not require IFN-R or STAT1 (Fig. 7C). To address whether the MyD88/TLR2/4-mediated differential regulation of TNF release and TNF mRNA induction applies to other cytokines, we determined IL-10 mRNA and protein amounts in Mtb-infected wt, MyD88–/–, and TLR2/TLR4–/– macrophages. As shown in Fig. 7, D and E, the expressions of IL-10 mRNA and protein were MyD88 and TLR2/4 independent. Thus, the differential involvement of TLR signaling in transcriptional and post-transcriptional control appears to be gene specific and does not apply to all cytokines.
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R or STAT1, for their induction
Mtb induces a group of genes in a manner that is completely or partially MyD88 dependent (7), including formyl peptide receptor, serum amyloid A3, macrophage receptor with collagenous domains, IL-6, and IL-1. Regulation of these genes in macrophages exposed to Mtb also required TLR2 or TLR4 and TIRAP, but not TRIF (Fig. 8A), IFN-R, or STAT1 (Fig. 8B; data for macrophage receptor with collagenous domains and IL-6 not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The present report reveals that MyD88-independent gene regulation in macrophages responding to viable, virulent Mtb is also independent of TLR2, TLR4, and the TLR3- and TLR4-serving adaptors, TIRAP and TRIF. Some genes whose regulation in response to Mtb meets the foregoing characteristics require, instead, IFN-R and STAT1 for their expression in Mtb-infected macrophages. Conversely, Mtb-induced, MyD88-dependent gene regulation for the most part requires TLR2 or TLR4, but neither IFN-R nor STAT1.
Live Mtb can activate cells that overexpress TLR2 or TLR4 (5, 37, 38). In contrast, using mouse bone marrow-derived DC isolated from TLR4-mutant or TLR2-deficient mice, Jang et al. (39) demonstrated that the absence of either TLR2 or TLR4 signaling had no impact on the ability of these cells to mature in response to infection by Mtb, as assessed by NF-
B activation and Th1-polarizing activity. However, lack of TLR2 diminished the production of TNF, IL-6, and IL-10 after Mtb infection (39). Blood cells from two individuals with homozygous IL-1R-associated kinase 4 deficiency produced subnormal amounts of TNF in response to viable Mtb (40). Both the studies by Jang et al. (39) in mice and by Picard et al. (40) in humans are consistent with our finding that TNF protein is produced by Mtb-infected macrophages in a MyD88- and TLR2/4-dependent manner. However, in both reports, transcriptional activation of TNF mRNA was not assessed. Our results point toward a differential role of the TLR/MyD88 pathway in mediating gene induction at the transcriptional level and in post-transcriptional control. TLRs signal through members of the MAPK family; post-transcriptional regulation of TNF may involve p38 MAPK-controlled TNF mRNA stability and/or translation (41, 42, 43). Similarly, IL-8 production is controlled post-transcriptionally via TLR5-mediated activation of p38 MAPK in epithelial cells (44).
Evidence bearing on the roles of TLR2 and TLR4 in host response to Mtb infection in mice has been ambiguous. Five independent studies have studied the susceptibility of TLR4-mutant C3H/HeJ mice to aerosol Mtb infection compared with a C3H strain with wt TLR4 alleles. Three studies showed that TLR4-defective mice were as resistant as congenic control mice after both low dose (100–350 CFU) and high dose (2000 CFU) Mtb infection (10, 11, 12). In contrast, two studies showed that TLR4-defective mice succumbed faster than control mice to low dose (50–100 CFU, aerosol) and high dose (105 CFU, intranasal) Mtb infection (8, 9). TLR2-deficient mice displayed reduced clearance of Mtb after aerosol infection with 100 and 500 live bacilli and succumbed within 5 mo after infection with 500 Mtb bacilli (45), whereas others found only a minor role of TLR2 in the control of Mtb (11, 46). Reiling et al. (11) showed that TLR2-deficient mice succumbed prematurely only to a high dose of Mtb (2000 CFU); when the mice were infected by aerosol with a lower dose (100 CFU), bacterial growth in lung, spleen, and liver and time to death were the same as in control mice. In our hands, after aerosol infection with either 50 or 600 CFU of live, virulent Mtb, TLR2/TLR4 double-knockout mice were as resistant as congenic control mice, as assessed by bacterial load, granuloma formation in the lungs, and host survival. Variables leading to the discrepant results among these studies remain to be identified, but a recent discovery may hold the key. TLR2 has been implicated in adrenal glucocorticoid regulation, and TLR2-deficient mice displayed an impaired stress response both at rest and during experimentally induced inflammation (47). Thus, conditions of animal husbandry, handling, and experimental protocol may differentially affect the level of immunoregulatory endocrine hormones that influence the response of TLR2-deficient mice to bacteria.
The role of MyD88 has also been ambiguous. In one study, MyD88–/– mice did not show increased mortality despite developing higher bacterial loads than wt mice (48). In two other studies, MyD88–/– mice failed to control mycobacterial replication and succumbed with a mean survival time of <4 wk (22, 23). In our hands, MyD88–/– mice were also highly susceptible to Mtb and died within 40 days after infection, consistent with the work by Scanga et al. (22) and Fremond et al. (23). The disparate results by Sugawara et al. (48) may be explained by the different bacterial strains used. That MyD88 played a major role both in our work and in the reports of others (22, 23) might implicate a key contribution of MyD88-dependent TLRs other than TLR2 and TLR4. However, there is more evidence to favor alternative explanations for MyD88’s prominent role in vivo that are not directly related to TLRs, such as transducing signals from IL-1R and IL-18R (49, 50, 51, 52, 53), priming macrophages to respond to IFN- (7), and controlling the stability of mRNAs for key immunoregulatory factors induced in response to IFN- (D. Sun and A. Ding, unpublished observations).
That TLR2 and TLR4 are collectively dispensable for host immunity against Mtb in the mouse is particularly surprising, inasmuch as certain gene products produced in response to Mtb in a TLR2/4- and MyD88-dependent manner, such as TNF, are of paramount importance to the host response in both experimental (54, 55) and clinical (56) tuberculosis. Most likely, this points to the key role of post-transcriptional regulation in TNF production, indirect routes to the induction of TNF in macrophages during Mtb infection in vivo, such as through CD40-CD40L interactions, and alternative sources of TNF besides macrophages, such as T cells.
The major conclusions of this work in the context of our earlier study (7) are that most likely there are receptors other than TLR2 and TLR4 that mediate the larger part of macrophage activation after Mtb infection, and that these receptors are not necessarily members of the TLR family. Recently, TLR2-, TLR4-, and MyD88-independent inhibition of IFN- responses by purified mycolylarabinogalactan-peptidoglycan in macrophages has been described (57). However, the receptor engaged by mycolylarabinogalactan-peptidoglycan remains unidentified. Moreover, our work suggests that these other receptors do not consist exclusively in one of the following: macrophage mannose receptor, CR3, CR4, SR-A, or CD40. However, we cannot exclude that two or more of the latter receptors may signal in an important and mutually redundant manner.
Mtb induced production of type I IFN has been shown in human DC and infected mouse lungs (58, 59). Secreted type I IFNs act in autocrine and paracrine fashions, and IFN- signaling in Mtb-infected macrophages and DCs is characterized by positive and negative regulations. In human DC, rapid production of IFN- in response to Mtb infection is followed by a delayed production of IFN-1 and/or IFN-13, suggesting positive feedback regulation (60). However, Mtb not only induces IFN-, it specifically inhibits IFN--mediated signaling via inhibiting STAT-1 activation in THP-1 cells and primary human macrophages (61). In contrast, Mycobacterium bovis Calmette-Guérin bacillus did not interfere with IFN- signaling, suggesting that the ability to inhibit type I IFN responses may be related to pathogenicity (61). Inhibition of IFN- signaling resulted from a negative feedback response to early secreted IFN- and required an additional, as yet unidentified, stimulus (62). Thus, the response to IFN- in Mtb-infected macrophages may be limited by Mtb’s ability to interfere with type I IFN signaling.
IFN-R–/– mice are less able than wt mice to control replication of Mtb after low dose aerosol infection (63). The mycobacterial titer in lungs from IFN-R–/– mice was increased at 2, 3, and 6 wk after infection, but was identical with that in wt mice at 12 wk after infection, suggesting that the activity of IFN- can be compensated by IFN- produced by activated T cells. Thus, type I IFN may be critical for the innate immune response to Mtb infection. In clinical studies a beneficial effect of treatment of tuberculosis patients with inhaled IFN- has been observed (64, 65), and people with STAT1 mutations are susceptible to mycobacterial infections, demonstrating a protective role of IFNs (66). In contrast, treatment of human monocytes with IFN- resulted in increased growth of M. bovis Calmette-Guérin bacillus, and intranasal instillation of IFN- in Mtb-infected mice increased mycobacterial growth and impaired mouse survival (59, 67). Thus, the roles of type I IFN for the pathogenesis of and protection against Mtb are complex and are likely to be beneficial as well as detrimental depending on the context. Similarly, type I IFNs played variable roles in innate immunity against other intracellular pathogens. IFN-R-deficient mice showed increased resistance to infection with Listeria monocytogenes, which appeared to be due to a release of IFN--mediated TNF- suppression and a block of IFN--induced apoptosis of splenic lymphocytes (68, 69, 70). In contrast, IFN- inhibited growth of intracellular Legionella pneumophilia in primary mouse macrophages via an IFN--independent, as yet unidentified, mechanism (71). IFN- was also found to increase resistance against Toxoplasma gondii, Leishmania major, and Clamydia psittaci (72, 73, 74). Taken together, the impact of type I IFN may vary and depend on several aspects of the host-pathogen interaction (75).
We demonstrated that Mtb induces IFN- mRNA expression in mouse macrophages and that the expression of immunologically relevant genes requires IFN-R and STAT1, but not TLR2/4, MyD88, TIRAP, or TRIF. Similar to our observation, expression of IP-10 in Mtb-infected human DCs was also dependent on IFN-, suggesting that chemokine responses in DCs are modulated by IFN- in an autocrine and paracrine manner (76). Induction of type I IFN via TLR7, TLR8, and TLR9 in DCs requires MyD88 (77, 78, 79, 80), in contrast to the pathway activated by Mtb. IFN- can also be induced in a TRIF-dependent manner by TLR4 and TLR3 ligands (27, 81). However, neither TLR4 nor TRIF was required for induction of IFN- mRNA by Mtb. Thus, none of the foregoing pathways appears to be essential for type I IFN induction by Mtb. Without testing TRIF/MyD88 double-deficient mice, we cannot rule out that the TLR4/TRIF pathway might be redundant with the TLR7/TLR8/TLR9/MyD88 pathway. However, this seems unlikely, because one would expect at least a partial loss of response in the single adaptor mutants, which we did not observe. Similar to our observations, Stockinger et al. (82) showed that macrophages infected with L. monocytogenes produced type I IFN via a pathway independent of TLR4, TLR9, MyD88, TRIF, TRIF-related adaptor molecule, and nucleotide-binding oligomerization domain 2.
In summary, we have provided evidence for four pathways leading to gene regulation in response to intact, viable Mtb in primary mouse macrophages. In pathway 1, Mtb signals through TLRs, chiefly TLR2, in a manner dependent on MyD88. In pathway 2, Mtb signals through TLR2, but not through MyD88; this pathway, although novel and mechanistically intriguing, is used sparingly. In pathway 3, Mtb signals through an unknown receptor(s), apparently leading as a primary response to production of type I IFN, because the transcriptional responses to Mtb appear to be secondary and depend, at least for the genes tested in this study, on IFN-R and STAT1. In pathway 4, Mtb transcriptionally activates gene expression in a TLR2/4-, MyD88-, IFN-R-independent manner, but protein production requires MyD88 and TLR2/4. It is the third pathway that dominates the response to viable Mtb quantitatively and physiologically in primary mouse macrophages, as judged by global gene expression analysis. Thus, efforts to seek the major signaling receptors of macrophages for intact, viable Mtb should continue.
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1 This work was supported by a Cancer Research Institute Predoctoral Fellowship Training Grant (to S.S.) and National Institutes of Health Grant HL68525 (to S.E.). The Department of Microbiology and Immunology is supported by the William Randolph Hearst Foundation. 
2 Address correspondence and reprint requests to Dr. Sabine Ehrt, Department of Microbiology and Immunology, Weill Cornell Medical College, Box 62, 1300 York Avenue, New York, NY 10021. E-mail address: sae2004{at}med.cornell.edu
3 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; BMM, bone marrow-derived macrophage; CR, complement receptor; DC, dendritic cell; iNOS, inducible NO synthase; IP10, IFN-inducible protein 10; MOI, multiplicity of infection; MR, mannose receptor; qRT-PCR, quantitative real-time PCR; SR-A, type A scavenger receptor; TIR, Toll-IL-1R; TIRAP, TIR domain-containing adapter protein; TRIF, TIR domain-containing adapter inducing IFN-; wt, wild type; IRG1, immune-responsive gene 1.
4 The online version of this article contains supplemental material.
Received for publication January 14, 2005. Accepted for publication June 25, 2005.
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, Genes regulated in wt and TLR2–/– macrophages; 
, genes whose regulation was dependent on TLR2. TLR2-dependent genes were defined as genes whose fold change in regulation in response to Mtb in TLR2–/– macrophages was statistically different (p 
, gene expression in lungs 21 days after Mtb infection. Data are the mean ± SD (n = 3 mice). Viable bacteria were assayed by counting CFU from lung homogenates plated onto enriched 7H11 plates 24 h, 14 days, 21 days, and 56 days after infection (B and D). An additional time point (140 days) was assayed after infection with 
