Published online before print January 13, 2006
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Institut fuer Mikrobiologie, Zentrum fuer Infektionsmedizin, Stiftung Tieraerztliche Hochschule Hannover, Germany
1 Correspondence: Institut fuer Mikrobiologie, Zentrum fuer Infektionsmedizin, Stiftung Tieraerztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. E-mail: ralph.goethe{at}tiho-hannover.de
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Key Words: mRNA stability • pathogenic and nonpathogenic mycobacteria • RAW264.7 • J774A.1 • p 38 • cycloheximide
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Macrophages are activated through the recognition of microbial products by germ line-encoded pattern recognition receptors (PRR), such as Toll–like receptors (TLR) [5 ]. The recognition of the various types of bacteria and their products induces common and specific signaling pathways, which, respectively, lead to the activation of different gene expression programs [6 7 8 ]. The induced program seems to result from the sum of the various types of microbial products expressed and their capacity to interact and activate different PRR on macrophages.
One well-known example of a microbial product with high activation capacity is the lipopolysaccharide (LPS) found in the outer membrane of gram-negative bacteria. LPS stimulates macrophages through a receptor complex that contains TLR4 as an essential central element [9 ], and it has been shown that the stimulation of macrophages with LPS and an infection with gram-negative bacteria induce similar changes in macrophage gene expression [10 ]. Thus, LPS can be considered as the predominant stimulating component of gram-negative bacteria and therefore, TLR4 signaling as the predominant activation pathway.
In contrast to gram-negative bacteria, mycobacteria seem to activate macrophages through various components, which act prevalently via TLR2. However, also, TLR4 and probably other TLR seem to be involved [11 12 ]. Conversely, pathogenic mycobacteria are able to impair cell signaling pathways required for cell activation [13 ].
The most important pathogenic mycobacterial species include Mycobacterium avium subspecies (ssp.), Mycobacterium bovis, Mycobacterium leprae, and Mycobacterium tuberculosis. M. avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne’s diseases in ruminants and is also associated with Crohn’s disease in humans [14 15 16 ]. MAP shows a strong tropism to the intestine, resulting in a chronic granulomatous enteritis in ruminants, which makes it unique within pathogenic mycobacteria.
We have previously shown that MAP induces cytokine expression typical for activated macrophages in murine J774A.1 macrophages. Conversely, MAP survives in these cells by inhibiting phagosomal acidification and phagosome lysosome fusion [17 18 ]. Furthermore, we could demonstrate that MAP survival in J774A.1 macrophages had no influence on the expression of major histocompatibility complex class II, intracellular adhesion molecule-1, B7.1, B7.2, or CD40. However, the antigen-specific stimulatory capacity of J774A.1 macrophages for a CD4+ T cell line was significantly inhibited after infection with viable MAP but not with viable M. avium ssp. avium (MAV) [17 ], indicating that murine macrophage cell lines are suitable models to detect differences in mycobacterial-induced effects up to the subspecies level.
Differential gene expression patterns in LPS-stimulated and mycobacteria-infected macrophages have been described [6 ]. However, the responsible molecular mechanisms have not been addressed. Even less is known about the molecular mechanisms that are responsible for the disruption of macrophage activation through infection with MAP.
In the present study, we compared mRNA expression responses in MAP-infected macrophages with that in LPS-treated macrophages using the representational difference analysis of cDNA. We could show that compared with LPS-stimulated macrophages, the expression levels of the immune-responsive gene 1 (IRG1) were significantly lower in MAP-infected macrophages. Furthermore, the IRG1 mRNA expression in Mycobacterium smegmatis-infected macrophages differed from that in LPS-stimulated as well as from that in MAP-infected macrophages. Transcriptional activation and mRNA stability were responsible for the differential of the IRG1 mRNA expression.
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32P]Deoxy-cytidine 5'-triphosphate (dCTP) and [32P]uridine 5'-triphosphate (UTP) were from Perkin-Elmer (Rodgau-Juegesheim, Germany). For plasmid labeling, the nick-translation kit from Life Technologies (Karlsruhe, Germany) was used.
Bacterial strains
The MAP strain DSM 44135 [19
] and the MAV strain (DSM 44156; ATCC 25291) were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). M. smegmatis mc2155 (ATCC 19420) was kindly provided by Dr. Georg Auling (Institut für Mikrobiologie, Universität Hannover, Germany). All mycobacteria were cultured as described previously [18
]. Escherichia coli Dh5 was used for propagating vectors in clones. E. coli were grown on Luria-Bertani (LB) medium supplemented with 100 µg/ml ampicillin at 37°C.
Plasmid construction
For cloning of the IRG1 promoter, a 984-base pair (bp) fragment of the 5' region of the IRG1 mouse genome sequence (AC L 38281) was generated by polymerase chain reaction (PCR) with primers, including cleaving sites for XhoI or HindIII at their ends (5'-CCGCTCGAGTCTCTGAGCCTTAGGTGTAGGG-3'; 5'-CCCAAGCTTTTGCTCTGGAGGGTAACTGG-3'). PCR was performed with genomic DNA from J774A.1 macrophages (1 µg) and 0.5 µM primers, 0.2 mM desoxyribonucleotides, 1.5 mM MgCl2, 1x PCR buffer, and 2.5 units Taq polymerase (Life Technologies, Gaithersburg, MD). Conditions were 91°C for 3 min followed by 25 cycles of 95°C for 1 min, 60°C for 2 min, and 72°C for 2 min, followed by a final extension at 72°C for 7 min. The resulting fragment was treated with XhoI or HindIII, ligated in the pGL3 basic vector (Promega), and transformed in E. coli Dh5. Insert sequences were controlled by sequencing (SeqLab, Göttingen, Germany). Endotoxin-free plasmids were prepared with the PureYieldTM plasmid midiprep system (Promega), according to the manufacturer’s protocol.
Macrophage cell culture
The murine macrophage-like cell lines RAW264.7 (ATCC, TIB-71) and J774A.1 cells (ATCC, TIB-67) were maintained in complete medium [Dulbecco’s modified Eagle’s medium, supplemented with 10% (v/v) fetal calf serum, 1% (w/v) L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies)] at 37°C in 75 cm2 flasks (Nunc, Roskilde, Denmark) in the presence of 8% CO2 in a humidified incubator. Bone marrow-derived macrophages (BMDM) from Balb/c mice, which have been differentiated according to the method of Lutz et al. [20
], were kindly provided by Wolfgang Bäumer (Institut fuer Pharmakologie, Toxikologie und Pharmazie, Stiftung Tierärztlichen Hochschule Hannover, Germany) and maintained as described above. For infection experiments, cells were seeded on 3.5 cm2 cell-culture dishes or six-well plates (Nunc). Twenty-four hours before infection, medium was removed, and after washing the monolayer with PBS, the cells were fed with fresh antibiotic-free medium. Then, cells were left untreated or stimulated with LPS (5 µg/ml). Infection of macrophages with mycobacteria was performed as described [18
]. Briefly, macrophages were incubated with a suspension containing prevalently single mycobacteria of an optical density at 660 nm (OD660) of 0.15 in antibiotic-free medium for 2 h. Then the medium was removed, and cells were washed with PBS and fed with fresh antibiotic-free medium. In some experiments, the macrophages were treated with latex beads (1 µm in diameter, Sigma), suspended at an OD660 of 0.15 for 2 h. In the titration experiments, macrophages were infected with mycobacteria of OD660 of 0.2, 0.1, 0.05, 0.025, and 0.0125, representing multiplicities of infection (MOI) of 
1:20, 1:10, 1:5, 1:2.5, and 1:1, respectively, as described above. For determination of RNA stability after infection with mycobacteria or stimulation with LPS, cells were treated with 5 µg/ml actinomycin D for up to 8 h. In some experiments, SB203580 (10 µM) was added simultaneously with actinomycin D. In some experiments, cells were treated with CHX (10 µg/ml) 10 min before stimulation or infection. For mycobacterial infection, CHX was added to the medium during and after infection.
RNA isolation and Northern analysis
Whole cell RNA was isolated using the RNeasyTM kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Poly(A)+ RNA preparation and Northern analysis were performed as described [21
]. Hybridization was performed with the [32P]-labeled plasmids pIRG or pGAP, containing the cDNA fragments from pIRG and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), respectively. After washing, the membranes were exposed to a Kodak Biomax film with intensifying screens at –70°C. To quantify hybridization signals, autoradiograms were analyzed densitometrically using an imaging documentation system from Bio-Rad (Munich, Germany).
Representational difference analysis of cDNA (cDNA RDA)
The cDNA RDA described here was based on the procedure described by Hubank and Schatz [22
] with some modifications. Briefly, cDNA RDA was performed with poly(A)+ RNA from J774A.1 macrophages, which have been stimulated with LPS for 24 h or infected with MAP for 24 h. Four micrograms was reverse-transcribed (RT) into cDNA with Superscript®II (Life Technologies), according to the manufacturer’s protocol. Tester and driver samples were prepared in parallel with cDNA from LPS-stimulated macrophages as tester and MAP-infected macrophages, as the driver and vice versa (LPS-MAP/MAP-LPS). Two micrograms of double-stranded cDNA was digested with 20 U DpnII, phenol/chloroform/isoamylalcohol (pci; 25:24:1)-extracted, and ethanol-precipitated and ligated to the first oligonucleotide RBGL12/24, as described by Braun et al. [23
]. After amplification [23
], the so-called amplicon was DpnII-digested, extracted with pci, preciptated with ethanol, and diluted in 300 µl H2O to generate the first driver. The first tester DNA was prepared by ligation of 1 µg purified driver DNA (concert rapid purification system, Life Technologies) to the second oligonucleotide IBGL12/24 as described. The first difference products (DP1) were produced by coprecipitation of 40 µg driver amplicons and 0.4 µg tester amplicons. After initial denaturation (85°C, 10 min), re-annealing was performed at 42°C for 24 h in buffer containing 40 mM piperazine-bis-2-ethane sulfonic acid, 1 mM EDTA (pH 8.0), 0.4 M NaCl, and 80% (v/v) formamide, followed by a precipitation to remove the formamide. Tester-specific PCRs were performed as described [22
]. Second and third subtractive hybridization was performed by coprecipitation of subtracted driver and subtracted tester in a ratio 400:1 and 4000:1. For detailed reaction conditions and oligonucleotide sequences, see Hubank and Schatz [22
]. Amplified subtraction samples were loaded on a 1.5% agarose Tris-borate-EDTA gel, which was stained with 0.5 µg/ml ethidium bromide (Sigma). Predominant bands were gel-eluted using the Geneclean III kit (Qbiogene, Heidelberg, Germany) and subcloned into pCR®II-TOPO vector (Life Technologies). Colony lifts were hybridized to the radiolabeled amplicons according to standard procedures [24
], and the plasmids from positive clones were dot-blotted and again hybridized to the radiolabeled amplicons. DNA sequencing was performed by automated means (SeqLab), and nucleic acid homology searches were performed using the Blast program at the European Molecular Biology Organization (Heidelberg, Germany) database.
RT-PCR
RT was performed with 200 ng poly(A)+ RNA using oligo(dT) primers and Superscript®II (Life Technologies) as described [17
]. PCR was performed using the following primers specific for ß-actin (5'-TGGAATCCTGTGGCATCCATGAAA-3'; 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'), GAPDH (5'-TGAAGGTCGGTGTGAACG-GATTTGGC-3'; 5'-CATGTAGGCCATGAGGTCCACCAC-3'), and IRG1 (5'-GGCTGTCCTACCTGTCCTCAC-3'; 5'-GGGCCTGTTTACGTACTGGAC-3').
The PCR conditions were 94°C for 3 min, 20 (ß-actin) and 21 (GAPDH) 25 (IRG1) cycles at 95°C for 1 min, 60°C for 1 min, 72°C for 2 min, followed by a final extension of 72°C for 5 min. PCR products were fractionated on 1.5% agarose gels at 75 V for 2.5 h, stained with ethidium bromide, and documented with a gel documentation system (Bio-Rad).
Transient transfection and luciferase assay
The day before transfection, RAW264.7 cells were plated at a cell density of 4 x 106 in 10 ml culture medium on cell-culture dishes (
94 mm) and incubated overnight to reach a confluence of 80%. Transfection of macrophages was performed with ExGene 500 (Fermentas), 0.75 µg of the indicated Firefly luciferase expression vector, and 0.025 µg Renilla luciferase (Promega), and an incubation for 6 h at 37°C. Following, medium was removed, and cells were washed twice with PBS, scraped off in serum-free medium (PAA, Coelbe, Germany), and split onto 12-well cell-culture plates. The next day, transfected cells were stimulated with LPS (5 µg/ml) or infected with mycobacteria for 24 h as described. Firefly and Renilla luciferase activities were measured in a luminometer (Lumat LB 9507, Berthold Technologies, Wildbad, Germany) using the dual luciferase assay system (Promega), as described by the manufacturer. Luciferase activity was normalized with Renilla luciferase activity and protein concentration and expressed as relative luciferase units.
Nuclear run-on transcription assay
Nuclear run-on transcription assays were performed according to the procedure described by Goethe and Phi-van [21
]. Briefly, 1.5 x 107 cells were suspended in 1 ml of a cell lysis buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.5% (v/v) Nonidet P-40 (NP-40) and incubated at 4°C for 5 min. Nuclei were harvested by centrifugation at 500 g for 5 min at 4°C and then washed once with 2 ml NP-40 lysis buffer. After centrifugation, nuclei were suspended in 100 ml of a storage buffer containing 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 0.1 mM EDTA, and 40% (v/v) glycerol and frozen at –80°C. For run-on transcription, nuclei were thawed on ice, immediately mixed with an equal volume of a run-on transcription buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, 0.5 mM each adenosine 5'-triphosphate, CTP, guanosine 5'-triphosphate, and 10 µCi [32P]UTP (800 Ci/mmol), and incubated for 30 min at 30°C. RNA was isolated by treatment with 200 U RNase-free DNase I for 15 min at 30°C, followed by incubation with 80 mg/ml proteinase K for 45 min at 37°C, extraction with Trizol® (Life Technologies), and two cycles of precipitation. Isolated DNA fragments containing the cDNA for GAPDH and IRG1 were denatured with 0.2 N NaOH for 10 min at 37°C and slot-blotted in the presence of 0.125x saline sodium citrate onto nylon membranes using a Hybri-Slot manifold (Life Technologies). After, ultraviolet-cross-linking filters were hybridized to the purified, labeled RNAs, according to the method developed by Church and Gilbert [25
]. To quantify hybridization signals, autoradiograms were analyzed densitometrically using an imaging documentation system from Bio-Rad.
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6 h, and expression decreased after 12 h (Fig. 2A
). It is interesting that the same kinetics of IRG1 mRNA expression was found in both macrophage cell lines after infection with MAP; however, at every time-point, the expression levels were considerably lower than in LPS-treated macrophages. Furthermore, semiquantitative RT-PCR analysis was performed with RNA from J774A.1 macrophages infected with MAP or MAV and from macrophages that had been treated with latex particles. As shown in Figure 2B
, both mycobacteria species induced IRG1 expression, whereas latex particles influenced IRG1 expression only marginally.
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Figure 1. Differential expression of IRG1 in LPS- and MAP-infected macrophages. (A) J774A.1 cells were stimulated with LPS (5 µg/ml) or infected with MAP for 24 h as described in Materials and Methods. Poly(A)+ RNA was isolated, and 4 µg was used for cDNA synthesis and RDA of cDNA. Agarose gel electrophoresis of DpnII-digested amplicons and DPs obtained after three subtraction hybridization-amplification steps from LPS-MAP (DP1–DP3) and MAP-LPS (DP1.1–DP1.3) are shown. (B) Four micrograms of poly(A)+ mRNA were used for agarose gel electrophoresis, transferred onto nylon membranes, and hybridized with [32P]-labeled probes specific for IRG1 and GAPDH. Untreated J774A.1 cells (control).
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Figure 2. Time kinetics of IRG1 mRNA expression in J774A.1 and RAW264.7, which (A) were stimulated with LPS (5 µg/ml) or infected with MAP for 0.5–48 h. Total RNA was isolated, electrophoresed, transferred onto nylon membranes, and subsequently hybridized with [32P]-labeled probes specific for IRG1 and GAPDH. (B) IRG1 gene expression in J774A.1 cells treated with latex beads or infected with MAP or MAV as determined by RT-PCR. J774A.1 cells were treated with latex beads or infected with MAP or MAV for 2–24 h. Poly(A)+ RNA was isolated, and 200 ng were used for cDNA synthesis. PCR was performed with primers specific for IRG1 and ß-actin and analyzed by gel electrophoresis.
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32P]UTP to elongate nascent mRNA transcripts. The labeled RNA was isolated and hybridized to an IRG1-specific cDNA fragment and as control, to cDNA specific for GAPDH immobilized on a nylon membrane. Figure 3
shows that the basal transcription rate of IRG1 was low in unstimulated cells and thus, consistent with the low RNA level shown in Figure 2A . After stimulation, the transcription rates increased 18-fold after 2 h and 14-fold after 6 h in LPS macrophages and approximately fourfold after 2 and 6 h in MAP-infected macrophages. As expected, the transcription rate of the GAPDH gene was not influenced in LPS-stimulated and MAP-infected macrophages. Thus, the results demonstrated that the IRG1 gene was regulated transcriptionally in LPS-stimulated and MAP-infected macrophages with approximately fourfold higher transcription rates in LPS-stimulated macrophages.
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Figure 3. The IRG1 gene is activated transcriptionally in LPS and MAP-stimulated RAW264.7 macrophages. Nuclei were isolated from untreated cells (–) or from cells activated with LPS or MAP for 2 h and 6 h and incubated in a run-on transcription assay with [-32P]UTP for 30 min to elongate RNA transcripts. RNAs were isolated, and equal amounts of radiolabeled RNAs were hybridized to cDNA fragments specific to IRG1 and GAPDH immobilized by slot-blotting onto nylon membranes.
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Figure 4. Increased IRG1 mRNA stability in RAW264.7 cells after stimulation with LPS. (A) Northern analyses of RNA from RAW264.7 cells, which had been infected with MAP or stimulated with LPS (5 µg/ml) for 16 h and were following treated with actinomycin D (ActD; 5 µg/ml) for 0.5–6 h. At the indicated time-points, total RNA was isolated. Twenty micrograms were used for agarose gel electrophoresis, transferred onto nylon membranes, which were hybridized subsequently with [32P]-labeled probes specific for IRG1 and GAPDH. (B) Percentage of mRNA remaining after treatment with actinomycin D. Values were calculated from the densitometric analysis of the IRG1 and GAPDH Northern blots shown in A. Open bars, Macrophages infected with MAP; solid bars, macrophages stimulated with LPS.
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Figure 5. IRG1 gene expression in RAW264.7 cells and BMDM from Balb/c mice stimulated with LPS or infected with MAP or M. smegmatis (MS). RAW264.7 cells were infected with MAP or M. smegmatis (A) or stimulated with MAP or MS or LPS (5 µg/ml; B), as described in Materials and Methods. Cells were lysed at the indicated time-points, and total RNA was isolated. Fifteen micrograms were used for agarose gel electrophoresis, transferred onto nylon membranes, and subsequently, hybridized with [32P]-labeled probes specific for IRG1 and GAPDH. (C) RNA was isolated from BMDM, which were infected with MAP or M. smegmatis or stimulated with LPS (5 µg/ml) for 8 h and analyzed for IRG1 expression by RT-PCR. (D) RNA was isolated from macrophages infected with increasing amounts of MAP or M. smegmatis (MOI) and analyzed for IRG1 expression as described above.
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Figure 6. IRG1 is stabilized transcriptionally and post-transcriptionally in M. smegmatis-infected RAW264.7. (A) cells were transfected with 0.75 µg luciferase reporter vector under control of the IRG1 promoter. After 24 h, cells were left untreated or treated with LPS (5 µg/ml) or infected with MAP or M. smegmatis for 24 h. Firefly luciferase activity was normalized to Renilla luciferase and to protein concentration. Results are expressed as fold of induction of the relative luciferase activities of untreated cells. Values are means ± SD of three independent experiments. (B) Northern blot analyses and diagrams illustrating decay of IRG1 mRNA in RAW264.7 cells, which were infected with MAP or M. smegmatis for 6 h and then treated with 5 µg/ml actinomycin D for 1–6 h. At the indicated time-points, total RNA was isolated, and 15 µg were used for agarose gel electrophoresis, transferred onto nylon membranes, and subsequently hybridized with [32P]-labeled probes specific for IRG1 and GAPDH (upper panel). The lower graphs shows the percentage of mRNA remaining after treatment with actinomycin D. Values were calculated as described for Figure 4
. Shaded bars, Macrophages infected with MAP; open bars, macrophages infected with M. smegmatis.
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(TNF-), a CHX-mediated mRNA increase was related to an increased mRNA stability [30
]. Therefore, mRNA stabilities in MAP and M. smegmatis-infected macrophages were compared with that of cells that had been treated with CHX before infection. As shown in Figure 7B
, inhibition of protein synthesis by CHX increased IRG1 mRNA stability in MAP and M. smegmatis-infected macrophages. Similar results were obtained with untreated cells (data not shown). As a result of the loss of cell viability, we could not determine the influence of CHX on IRG1 stability in LPS-stimulated cells. These results imply that a constitutively expressed destabilizing factor contributes to IRG1 mRNA stability.
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Figure 7. IRG1 mRNA stabilization is p38 MAPK-independent and increased by CHX. (A) Northern blot analyses (upper) and diagrams (lower) illustrating decay of IRG1 mRNA from RAW264.7 cells that had been infected with MAP or stimulated with LPS (5 µg/ml) for 6 h and following, treated with actinomycin D (5 µg/ml) or with actinomycin D and 10 µM SB203580 for 0.5–6 h. Total RNA was isolated, and 15 µg were used for agarose gel electrophoresis. RNA was transferred onto nylon membranes and subsequently hybridized with [32P]-labeled cDNAs for IRG1 and GAPDH (upper). Lower graphs show the percentage of mRNA remaining after treatment with actinomycin D. Values were calculated as described for Figure 4
. Solid bars, Without SB203580; open bars, with SB203580. (B) Northern analyses of RNA from RAW264.7 cells, which were left untreated or were treated with 10 µg/ml CHX for 15 min. Then, the cells were infected with MAP or M. smegmatis and after 6 h, treated with 5 µg/ml actinomycin D for 1 h and 4 h. For the following steps, see A. Lower graphs show the percentage of mRNA remaining after treatment with actinomycin D. Values were calculated as described for Figure 4
. Solid bars, Without CHX; open bars, with CHX.
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Nevertheless, we found that expression levels of the IRG1 gene were significantly higher in LPS-stimulated than in MAP-infected macrophages. The IRG1 gene was first identified by Lee et al. [26 ] as a novel 2.3-kb cDNA, which was obtained from a cDNA library prepared from LPS-stimulated RAW264.7 cells. By interspecific back-cross analysis, IRG1 was mapped to mouse chromosome 14 linked to Tyrp2 and Rap2a. An open reading frame analysis of its sequence showed that it is highly conserved in vertebrates and that it has high homology to the bacterial protein methlycitrate dehydrogenase. This enzyme is involved in the dehydration of (2S,3S)-methylcitrate to 2-methylcisaconitate [32 33 ]. The high homology of IRG1 protein sequences among mammals suggests that it may have an indispensable role in metabolism. Its role in macrophages is unknown.
Several studies about TLR signaling and their adaptors in LPS-stimulated and M. tuberculosis-infected macrophages identified a peculiar regulation of the IRG1 gene. Studies about murine DC and macrophages have shown that the LPS-induced IRG1 gene expression was dependent on TLR4 in a myeloid differentiation primary-response protein 88 (MyD88)- and interferon-ß (IFN-ß)-independent manner [34 35 ]. In a more recent study with LPS-stimulated macrophages from MyD88–/– and Toll/interleukin-1 receptor (IL-1R) domain-containing adaptor-inducing IFN-ß (TRIF–/–)-deficient mice, Hirotani et al. [36 ] observed that the IRG1 gene was induced in MyD88–/– and TRIF–/– macrophages but unresponsive in MyD88–/–TRIF–/– macrophages, indicating that the LPS-induced IRG1 expression was dependent on MyD88 and TRIF [36 ]. It is interesting that most recently, Shi et al. [27 37 ] showed that in M. tuberculosis-infected macrophage, IRG1 mRNA expression was independent from TLR2 and -4 and their combination as well as from MyD88, Toll/IL-1R domain-containing adapter protein, and TRIF. Furthermore, mycobacterial induction of IRG1 mRNA expression was observed in macrophages lacking mannose receptor, complement receptor 3, type A scavenger receptor, and CD40. None of these studies, however, considered the down-stream molecular mechanisms that contributed to the induced IRG1 mRNA expression.
To gain insights in the mechanisms of differential IRG1 expression, we first analyzed the time kinetics of IRG1 mRNA expression in LPS-stimulated and MAP-infected J774A.1 macrophages and compared it with RAW264.7 macrophages, another murine macrophage cell line. Both cell lines induced nearly the same expression kinetics after LPS stimulation and after infection with MAP; however, at any time-point, the expression was notably lower in MAP-infected macrophages. This difference seemed not to be solely a result of increased transcription rates, as our run on transcription analyses revealed only an approximately fourfold higher transcription rate in LPS-stimulated than in MAP-infected macrophages.
Inflammatory gene expression is tightly regulated at the transcriptional and post-transcriptional levels [28 ]. Our Northern analysis showed that IRG1 mRNA stability was approximately two- to threefold higher in LPS-stimulated than in MAP-infected macrophages. Thus, transcriptional activation and mRNA stabilization seem to be responsible for differential IRG1 expression in LPS-stimulated and MAP-infected macrophages. Macrophages infected with pathogenic MAV behaved similar as those infected with MAP, as demonstrated by our RT-PCR analysis and Northern analysis (data not shown). Controls with latex beads showed that phagocytosis seemed not to induce IRG1 expression, indicating that IRG1 gene induction depended on specific bacterial components.
We have previously shown that in contrast to pathogenic MAP, nonpathogenic M. smegmatis did not survive in murine macrophages and did not inhibit phagosomal acidification and phagosome lysosome fusion [18 ]. One most impressive finding of our present study was that IRG1 mRNA expression in M. smegmatis-infected macrophages differed from that in LPS-stimulated as well as from that in MAP-infected macrophages. Thus, in M. smegmatis and MAP-infected macrophages, IRG1 mRNA transcription seemed to be similar at 2 h after infection. However, in the time course of infection, the IRG1 mRNA levels increased as a result of message stabilization, as in LPS-stimulated macrophages, and reached nearly the LPS-induced mRNA levels. Infection of macrophages with heat-inactivated M. smegmatis or MAP resulted in IRG1 stabilities similar as in macrophages infected with viable mycobacteria (data not shown). Thus, it is most likely that heat-stable, structural components of the mycobacteria contribute to the differential IRG1 mRNA stabilization in macrophages.
Increased mRNA stabilities have been described after stimulation of macrophages, including RAW264.7 cells, with LPS [38 39 40 ]. AREs within the 3'-UTRs of transcripts accelerate their decay [28 29 ]. The 3'-UTR of the IRG1 contains a conserved nonamer UUAUUUAUU motif [26 ], thus comprising a minimum functionally ARE UUAUUUA(A/U)(A/U) motif, which forms the optimal binding site for RNA destabilizing factors [41 42 43 44 ]. This motif is similar for ARE class II, although typical AREs class II contain at least two overlapping copies of the nonamer [29 ]. It has been shown that ARE class II-directed mRNA stabilization is dependent on the MAPK p38 [28 ]. In addition, several mRNAs, which do not contain typical AREs class II in their 3'-UTR, have been shown to be p38-regulated [45 46 ]. Furthermore, recent studies have shown a differential activation of the MAPKs in primary BMDM following infection with pathogenic MAV compared with the activation following infection with nonpathogenic M. smegmatis. Thus, diminished activation of p38 and extracellular signal-regulated kinase 1/2 was observed in macrophages infected with pathogenic strains of MAV compared with infections with nonpathogenic M. smegmatis [47 48 ]. Our results demonstrated that in LPS-stimulated and MAP-infected macrophages, the IRG1 mRNA stability was not affected by SB203580, a specific inhibitor of p38 MAPK, suggesting that IRG1 mRNA stabilization was p38 MAPK-independent.
Lee at al. [26
] showed that the IRG1 gene is induced after treatment of RAW264.7 macrophages with the protein synthesis inhibitor CHX. We show that treatment of RAW264.7 macrophages with CHX before infection induced IRG1 mRNA expression and increased IRG1 mRNA stability in M. smegmatis and MAP-infected macrophages. As shown for TNF- [30
], the CHX-mediated mRNA increase seemed to be related in part to an increased mRNA stability. These results suggest that a constitutively expressed destabilizing factor contributes to IRG1 mRNA stability. Stabilization of M. smegmatis-infected macrophages was not as pronounced as in MAP-infected macrophages. Furthermore, superinduction of IRG1 mRNA expression was not inducible in LPS-stimulated macrophages (data not shown and ref. [26
]). Hence, in LPS-stimulated and M. smegmatis-infected macrophages, IRG1 mRNA destabilization might be antagonized by inducible, stabilizing factors.
In conclusion, our results indicated that the regulation of IRG1 mRNA expression in macrophages differs at the transcriptional and the post-transcriptional level, dependent on the stimulatory agent. Although the function of IRG1 in macrophages is not known yet, we think that the IRG1 gene represents a suitable marker gene to dissect the molecular mechanisms contributing to macrophage activation by mycobacteria. The factors contributing to IRG1 mRNA expression and mRNA stabilization in RAW264.7 macrophages, however, remain to be identified.
Received September 15, 2005; revised November 21, 2005; accepted November 25, 2005.
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yet mediates few responses to Mycobacterium tuberculosis J. Exp. Med. 198,987-997}{ß} Receptor and STAT1 J. Immunol. 175,3318-3328 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade FEBS Lett. 483,57-61[CrossRef][Medline] and bacterial lipopolysaccharide. Transcriptional and post-transcriptional regulation J. Biol. Chem. 269,8324-8333 mRNA J. Biol. Chem. 273,13781-13786This article has been cited by other articles:
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