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Laboratories of
* Immunology,
Bacteriology and Medical Mycology, and
Virology, Istituto Superiore di Sanità, Rome, Italy;
Laboratory of Cytokine Signaling, Institut Pasteur, Paris, France; and
¶ Institute of Molecular Genetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5124, Montpellier, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. In this work we have
monitored a rapid induction of IFN-
followed by the delayed
production of the IFN-1 and/or -13 subtypes. The Mtb infection
rapidly activates the NF-
B complex and stimulates the
phosphorylation of IFN regulatory factor (IRF)-3, events known to
induce IFN- expression in viral infection. In turn, the autocrine
production of IFN- induces the IFN-stimulated genes that contain
binding sites for activated STATs in their promoters. Among the
IFN-stimulated genes induced in DC through STAT activation are IRF-1
and IRF-7. The expression of IRF-1 appears to be dependent on the
sequential activation of NF-B and STAT-1. Once expressed, IRF-1 may
further stimulate the transcription of IFN-. Induction
of IRF-7 is also regulated at the transcriptional level through the
binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated
gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears
to be required for the delayed induction of the IFN-1/13
genes. Although correlative, our results strongly support the
existence of a cascade of molecular events in Mtb-infected DC. Upon
infection, constitutively expressed NF-B and IRF-3 are activated and
likely contribute to the rapid IFN- expression. In turn,
IFN--induced IRF-1 and IRF-7 may cooperate toward induction of
IFN-1/13 if infection persists and these factors are
activated. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) is secreted by virtually
all cells in response to viral infection, type II (IFN-
) is mainly
produced as a result of stimulation of T lymphocytes and NK cells
(1). In line with the different cell origin, type I and II
IFN mediate diverse functions, including antiviral and antigrowth
effects and immunomodulatory activities (1). Although
IFN- possesses antiviral activity, it primarily promotes
antimicrobial and antitumor activities in macrophages (2).
Conversely, type I IFN, which are potent antiviral agents, have been
found to regulate the immune system through the control of the
proliferation, differentiation, activation, and maturation of different
leukocytic populations, such as dendritic cells
(DC),3 NK cells, Th1
cells, and memory CD8+ lymphocytes
(3, 4, 5, 6). Several immunoregulatory effects are shared by all
IFN. Indeed, type I and type II IFN induce overlapping but not
identical sets of genes via different cell surface receptors
(7). A major immunomodulatory role of IFN is exerted on
the generation, transport, and presentation of antigenic peptides
displayed on MHC class I (8, 9, 10, 11). Conversely, other
effects appear to be induced uniquely by IFN-, such as the MHC class
II proteins, or by IFN-, like the 2 subunit of the IL-12R on
human T cells (9). Recent studies have highlighted the production of type I IFN following bacterial infections (12, 13). It has been shown that the infection of THP-1 cells with Mycobacterium tuberculosis (Mtb) leads to secretion of type I IFN (13), and we have recently reported on the ability of Mtb-infected DC to produce type I IFN (14). Moreover, Cella et al. (15) found that plasmacytoid DC produce type I IFN in Mtb-infected lymph nodes. Despite these converging data, the mechanisms that regulate type I IFN expression in Mtb-infected cells are poorly defined.
In humans, all the known intronless type I IFN genes are clustered on
the short arm of chromosome 9 spanning 400 kb (16). While
IFN- is encoded by a single gene, IFN- are
encoded by a large family of related genes composed of 12 functional
IFN- and a single IFN-
. The expression of the IFN- genes
appears coordinately induced in virus-infected cells and may differ
among cell types, reflecting the transcriptional activity of the
corresponding promoter regions (17). A virus-responsive
element (VRE) has been identified in the IFN-1 gene
promoter (VRE-A1) that contains a positive regulatory domain
(PRD)I-like sequences, thus suggesting the involvement of IFN
regulatory factor (IRF) proteins (18, 19, 20, 21). The regulation
of the IFN- gene has been studied extensively. The VRE of
IFN- is composed of positive and negative regulatory
elements (22). The PRDIV binds a heterodimer of activating
transcription factor-2 and c-Jun, whereas PRDII binds NF-B. The PRDI
and III elements are known to bind specifically members of the IRF
family. Among the nine members of this family, IRF-1, -3, -7, and -9
have been identified as positive regulators of IFN- genes
(23, 24). Although IRF-1 was first identified by its
ability to bind the hexamer (AAGTGA) repeats in the IFN-
promoter and to induce transcription, the analysis of mice null for
this gene showed that IRF-1 is dispensable for IFN gene expression
(25). Evidence has been provided for the involvement of
the IFN-stimulated gene factor (ISGF)-3, a heterotrimeric complex
consisting of IRF-9, STAT-1, and STAT-2, in the IFN- expression
(26, 27, 28). More recently, it was found that IRF-3 and IRF-7
are involved in IFN- induction (29). IRF-3 undergoes
phosphorylation and nuclear translocation following virus infection
(30). This phosphorylation leads to the association of
IRF-3 with the general coactivator p300/CREB-binding protein and
the binding to the IFN- PRDI element
(31, 32, 33, 34, 35). While IRF-3 is constitutively expressed in
growing cells, IRF-7 expression is induced by IFN- through the
activation of ISGF-3 (36). Similar to IRF-3, IRF-7
undergoes virus-induced serine phosphorylation and nuclear
translocation (33, 36, 37, 38).
Once secreted from infected cells, IFN induce in an autocrine and paracrine fashion the activation of Janus kinases and STAT transcription factors, leading to expression of target genes (39). Tyrosine phosphorylation of STAT-1 and STAT-2 occurs in most cell types (40), although activation of STAT-3, -4, -5, and -6 has also been reported (41, 42, 43, 44). Upon phosphorylation, dimerization, and translocation into the nucleus, STATs bind to STAT-binding element (SBE) or to the IFN-stimulated response element (ISRE) in the promoter of target genes. Interestingly, several primary response genes are themselves transcription factors that are required for induction of secondary effectors of the cellular response to IFNs and to other cytokines. Among these, IRF-1 and IRF-7 are transcriptionally regulated by SBE and ISRE sites present within their respective promoters. Thus, the response to IFN as well as its production can be amplified and potentiated through the enhanced expression of IRF factors.
Mtb is an intracellular pathogen that infects human macrophages and DC
(14, 45, 46). The outcome of the infection is dependent
upon both innate and acquired immune responses (47, 48).
We have previously shown that the infection of DC with Mtb induces the
expression of markers involved in Ag presentation and T cell
interaction. Moreover, a typical pattern of Th1/IFN--inducing
cytokine production is elicited in infected DC that secrete IL-12 and
IFN- (14). In this work we have extended our studies to
identify which IFN subtypes are produced in Mtb-infected DC, and we
have investigated the molecular mechanisms of their regulated
expression. A cascade of events activates transcription factors,
previously shown to regulate IFN- expression in virally infected
cells. We report on the sequential expression of the IFN-
and IFN-1 or -13 genes in DC infected with
Mtb through the recruitment of IRF-1, -3, and -7 transcription
factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC were prepared as previously described (14).
Briefly, PBMCs were isolated from freshly collected buffy coats
obtained from healthy voluntary blood donors (Blood Bank of University
La Sapienza, Rome, Italy) by density gradient centrifugation using
Lympholyte-H (Cedarlane Laboratories, Hornby, Ontario, Canada).
Monocytes were purified by positive sorting using
anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch
Gladbach, Germany). The recovered cells were >99%
CD14+ as determined by flow cytometry with
anti-CD14 Ab. DC were generated by culturing adherent monocytes in
six-well tissue culture plates (Costar, Cambridge, MA) with 25 ng/ml
GM-CSF and 1000 U/ml IL-4 (R&D Systems, Abingdon, U.K.) for 5 days at
0.5 x 106 cells/ml in RPMI with supplements
as above. No antibiotics were added to the cultures. At day 5, the
cells were analyzed for the expression of surface markers associated
with DC differentiation. Resulting cells were 70–80%
CD1a+ and 95% CD14-. DC
were infected with Mtb at a Mtb/cell multiplicity of infection of 5, as
previously described (14). Where indicated, IFN-2
(recombinant human IFN-2b, provided by D. Gewert, QT Genetics,
Cambridge, U.K.) was used at 0.1 nM to monitor STAT activation.
Rabbit polyclonal antiserum raised against IFN- and - was used at
20 µg/ml (PBL Biomedical Laboratories, New Brunswick, NJ).
IFN determination
Supernatants from control and Mtb-infected DC cultures were
harvested at different times after infection, filtered (0.2-µm
filters), and stored at -80°C. Ab pairs used in ELISA for IFN-
and IFN- were from PBL Biomedical Laboratories. Supernatants from
6–10 separate experiments were considered. The ELISA were conducted
according to the manufacturer’s instructions.
Cell extracts
Nuclear cell extracts were prepared as described by Schreiber et al. (49). Briefly, cell pellets (5 x 106) were resuspended in 1 ml of buffer A (0.5% Nonidet P-40, 10 mM EDTA, 10 mM EGTA, 10 mM KCl, 10 mM HEPES (pH 7.9)) to which 1 mM DTT, 0.5 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml trypsin inhibitor, and 1 µg/ml antipain were freshly added) and incubated on ice for 10 min. Nuclei were sedimented by centrifuging the lysates at 1,200 x g for 10 min. The nuclear pellets were resuspended in 30–40 µl with buffer C (1 mM EDTA, 1 mM EGTA, 0.4 M NaCl, 20 mM HEPES (pH 7.9), 5 mM MgCl2, 25% glycerol, with fresh addition as above) and incubated for 10 min on ice with occasional mixing. The suspensions were clarified by centrifuging at 15,000 x g for 10 min. The supernatants were recovered as nuclear extracts and were rapidly frozen on crushed dry ice and stored at -80°C. Whole cell extracts were prepared as previously described (50). Briefly, cells (107) were lysed in 30–50 µl of ice-cold whole cell extraction buffer (20 mM HEPES (pH 7.9), 50 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 10 mM EDTA, 2 mM EGTA, 10 µg/ml leupeptin, 100 mM NaF, 0.5 mM PMSF, 10 mM sodium orthovanadate, and sodium molybdate). The lysate was incubated for 30 min on a shaker at 4°C and insoluble debris was removed by centrifugation (13,000 x g at 4°C for 10 min) and the lysate was stored at -80°C. Both whole and nuclear extracts from cells infected by Mtb were filter-sterilized before removal from BSL-3 containment.
EMSA
To measure the association of DNA-binding proteins with
different DNA sequences, synthetic double-stranded oligonucleotides
were end-labeled with [-32P]ATP by T4
polynucleotide kinase. For the analysis of NF-B complexes, nuclear
cell lysates (5 µg) were used in EMSA experiments. Binding reaction
mixture (20 µl final volume) contained labeled oligonucleotide probes
(30,000 cpm) in binding buffer (4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM
Tris-HCl (pH 7.5), 1 µg poly(dI)-poly(dC)). Nuclear lysates were
added and the reaction mixture was incubated for 30 min at room
temperature. For supershift analysis, 1 µg of anti-p50 (Santa
Cruz Biotechnology, Santa Cruz, CA) was added to the reaction. For the
analysis of IRF and STAT complexes, whole cell lysates (15 µg) were
used in EMSA experiments. The labeled oligonucleotide probe (30,000
cpm) was mixed with 2 µg of poly(dI)-poly(dC) (Amersham Pharmacia
Biotech, Little Chalfont, U.K.) and 1 µg of BSA
(Sigma-Aldrich, St. Louis, MO), and incubated for 30 min at 20°C in a
final volume of 20 µl of binding buffer (75 mM KCl, 20 mM Tris-HCl
(pH 7.5), 1 mM DTT) containing 15 µg of cell extract. For Ab
treatments, 1 µg of anti-STAT-1, STAT-2, and IRF-1 (Santa Cruz
Biotechnology) were added to the reaction mixture. Glycerol was added
to 13% (v/v) and samples were analyzed on 5% polyacrylamide gels with
0.5x TBE (1x TBE is 50 mM Tris-borate (pH 8.2) and 1 mM EDTA) for
1.5 h at 200 V at 18°C.
The oligonucleotides used were as follows: IFN- B
(5'-AGTGGGAAATTCCTCT-3') (22); IFN- PRDIII-I
(5'-GAAAACTGAAAGGGAGAAGTGAAAGTG-3') (22); IRF-1 B
(5'-GGGCCGGGGAATCCCGCTAAGT-3') (51); IRF-1 SBE
(5'-GATCGATTTCCCCGAAATGA-3') (52); IRF-7 ISRE
(5'-TTTAGGTTTCGCTTTCCCGG-3') (53); and IFN-1 VRE
(5'-GAGTGCATGAAGGAAAGCAAAAACAGAAATGGAAAGTGG-3')
(54).
DNA affinity purification assay
Biotinylated oligonucleotides wild-type IFN-1 VRE
(5'-GAGTGCATGAAGGAAAGCAAAAACAGAAATGGAAAGTGG-3'), and mutated
IFN-1 VRE (5'-GAGTGCATGAAGGACCGCAACAGCCATGGTTAGTGGCCCAGAAGCA-3')
were annealed with the corresponding antisense oligonucleotide in 1x
STE buffer, containing 10 mM Tris-HCl (pH 8), 50 mM NaCl, and 2 mM
EDTA. Biotinylated DNA oligonucleotides were mixed with 200 µg of
total extract in 500 µl of binding buffer containing 20 mM Tris-HCl
(pH 7.5), 75 mM KCl, 1 mM DTT, and 5 µg/ml BSA in presence of 13%
glycerol and 20 µg of poly(dI-dC) and incubated for 25 min at room
temperature. Then streptavidin magnetic beads (Promega, Madison, WI),
washed three times with 800 µl of binding buffer, were added to the
reaction mixture and incubated for 30 min at 4°C and for 10 min at
room temperature with mixing by rotation. The beads were collected with
a magnet and washed three times with 1 ml of binding buffer. The bound
proteins were eluted from the beads by boiling in sample buffer and
were resolved on 10% SDS-PAGE followed by immunoblotting with Ab
against IRF-1, IRF-3, and IRF-7.
Western blot analysis
Whole cell extracts (30 µg) were separated by 7 or 10% SDS-PAGE gel and blotted onto nitrocellulose membranes. Blots were incubated with rabbit polyclonal Abs against IRF-1, IRF-3, IRF-7, STAT-1, and STAT-2 (Santa Cruz Biotechnology) and reacted with anti-rabbit HRP-coupled secondary Ab (Amersham Pharmacia Biotech) using an ECL system. Tyrosine-phosphorylated STAT-1 and STAT-2 were detected by immunoblotting with phosphospecific Abs and, after stripping, with the specific STAT Abs to evaluate loading. Anti-phospho-STAT-1 Abs were from New England Biolabs (Beverly, MA). Anti-phospho-STAT-2 was a generous gift of Dr. D. Levy (New York University School of Medicine, New York, NY).
RNA isolation and real-time PCR quantification
RNA was extracted from DC with RNeasy kit (Qiagen, Valencia, CA)
according to the manufacturer’s instructions, which include a DNaseI
treatment. A phenol/chloroform extraction was performed to inactivate
residual mycobacterial particles. Reverse transcription were primed
with oligo(dT) and performed using the murine leukemia virus
reverse transcriptase from Life Technologies (Rockville, MD).
Quantitative PCR assays were done using the SYBR Green I technology on
a Light Cycler (Roche Diagnostic Systems, Somerville, NJ). The primer
pairs used were as follows: IFN-1/13 forward,
5'-TGGCTGTGAAGAAATACTTCCG-3'; IFN-1/13 reverse,
5'-TGTTTTCATGTTGGACCAGATG-3'; IFN-2 forward,
5'-CCTGATGAAGGAGGACTCCATT-3'; IFN-2 reverse,
5'-AAAAAGGTGAGCTGGCATACG-3'; IFN-4 forward,
5'-GAAGAGACTCCCCTGATGAATGT-3'; IFN-4 reverse,
5'-GCACAGGTATACACCAAGCTTCTTC-3'; IFN-5 forward,
5'-TCCTCTGATGAATGTGGACTCT-3'; IFN-5 reverse,
5'-GTACTAGTCAATGAGAATCATTTCG-3'; IFN-6 forward,
5'-CTGTCCTCCATGAGGTGATT-3'; IFN-6 reverse,
5'-GGTCTTATTCCTTCCTCCTTAAC-3'; IFN-7 forward,
5'-CAGACATGAATTCAGATTCCCA-3'; IFN-7 reverse,
5'-TTTCCTCACAGCCAGGATGA-3'; IFN-8 forward,
5'-GTGATAGAGTCTCCCCTGATGTAC-3'; IFN-8 reverse,
5'-CTTCAATCTTTTTTGCAAGTTGA-3'; IFN-10 forward,
5'-TGGCCCTGTCCTTTTCTTTACTT-3'; IFN-10 reverse,
5'-TCAAACTCCTCCTGGGGGAT-3'; IFN-14 forward,
5'-TGAATTTCCCCAGGAGGAA-3'; IFN-14 reverse,
5'-TCCCAAGCAGCAGATGAGTT-3'; IFN-16 forward,
5'-CAAAGAATCACTCTTTATCTGATGG-3'; IFN-16 reverse,
5'-CAATGAGGATCATTTCCATGTTGAAT-3'; IFN-17 forward,
5'-TGTGATACAGGAGGTTGGGA-3'; IFN-17 reverse,
5'-GTTTTCAATCCTTCCTCCTTAATA-3'; IFN-21 forward,
5'-ATCTCAAGTAGCCTAGCAATATTG-3'; IFN-21 reverse,
5'-AGGTCATTCAGCTGCTGGTT-3'; IFN- forward,
5'-GTCTCCTCCAAATTGCTCTC-3'; IFN- reverse,
5'-ACAGGAGCTTCTGACACTGA-3'; IFN- forward,
5'-GAGGTACTTCCAGGGAATCCG-3'; IFN- reverse,
5'-CATTTCAAGATGAGCCCAGGTC-3'; IRF-7 forward,
5'-GAGCCCTTACCTCCCCTGTTAT-3'; IRF-7 reverse,
5'-CCACTGCAGCCCCTCATAG-3'; GAPDH forward, 5'-ACAGTCCATGCCATCACTGCC-3';
GAPDH reverse, 5'-GCCTGCTTCACCACCTTCTTG-3'. The absence of
contaminating genomic DNA in reverse transcription products was
demonstrated by specific primer pairs for IFN- subtypes that do not
generate PCR products.
For GAPDH and IRF-7 quantification, standard curves were obtained using PCR products diluted in 10 µg/ml sonicated salmon sperm DNA. For type I IFN quantification, human genomic DNA (10–0.1 µg/ml) was used to generate the standard curves. The identity of PCR products were determined by DNA sequencing to validate the specificity of the primer pairs used and by melting curve analysis in all experiments. All data are presented as a ratio with GAPDH (95% confidence limits).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and - are expressed with different kinetics in
Mtb-infected DC
We have previously shown that DC infected with Mtb produce IFN-
(14). To identify the expression profile of type I IFN and
to investigate the kinetics of production, cell culture supernatants
were collected at different time points after Mtb infection and the
levels of IFN- and - were determined by ELISA (Fig. 1
A). DC infected with Mtb
produced both IFN- and IFN- subtypes, but with different
kinetics: IFN- was maximally detected at 16 h after infection,
while IFN- steadily increased from 24 h postinfection.
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subtypes, IFN-, and
IFN- was then studied in DC infected for 24 h with Mtb. For
this, mRNA levels were quantified by real-time RT-PCR using primers
specific for sequences within the coding region of each isotype.
Surprisingly, Mtb-infected cells did not induce a mixture of isotypes,
but induced mainly IFN-1 and/or IFN-13 (Fig. 1
B).
These two nonallelic genes differ only in few nucleotides in the 5' and
3' noncoding regions and their mRNAs give rise to the same protein
(55). To compare the kinetics of IFN- and
IFN-1/13 gene expression, RNA was isolated from DC at
different times after Mtb infection and the steady-state mRNA levels
were measured by real-time RT-PCR (Fig. 1C). At 16 h
postinfection, a clear correlation was observed between the secretion
of IFN- and the up-regulation of its transcript. The expression of
the IFN-1/13 mRNA was delayed, being induced 16 h after
exposure of the DC to Mtb and increasing up to 24 h. At 43 h,
the level of both IFN- and IFN-1/13 mRNAs decreased sharply.
Infection of DC with Mtb induces the activation of NF-B and IRF
transcription factors
NF-B is a major player in the transcription of the
IFN- gene. In particular, the p50/p65 heterodimer is
known to bind to the -66 to -57 region of the IFN-
promoter, termed PRDII (56, 57, 58, 59). To study whether Mtb
infection was able to induce binding of NF-B to the PRDII regulatory
sequence, DC were stimulated with live bacteria. Nuclear extracts were
prepared at different times and analyzed by EMSA using an
oligonucleotide corresponding to the B site of the
IFN- promoter (Fig. 2A). NF-B DNA binding was
detected at 1 h after infection and reached a maximal level at
8 h. The identity of the complex was confirmed by supershift
experiments using Abs raised against the p50 subunit.
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promoter contains also the PRDI and III elements
that are known to bind different IRF family members. We thus
investigated whether infection of DC by Mtb would induce IRF
DNA-binding activity as measured by EMSA using an oligonucleotide
corresponding to the PRDIII-I (Fig. 2B). A clear induction
of IRF-1 DNA binding activity was observed as early as 3 h
postinfection and a sustained activity was still present at 24 h.
The identity of the complexes was confirmed by reaction with
anti-IRF-1 Ab. This activation well correlated with the induction of
the IRF-1 protein (see Fig. 4B). Because we were unable to
detect IRF-3 binding activity by EMSA profile, we investigated whether
Mtb infection could induce IRF-3 phosphorylation, as demonstrated in
the event of virus infection (60, 61). Immunoblots were
performed on total cell extracts prepared at different times following
infection (Fig. 2C). As expected, IRF-3 was constitutively
expressed in uninfected cells in its nonphosphorylated form. Mtb
infection resulted in an evident increase of IRF-3 phosphorylation,
monitored by the appearance of slower migrating forms in 7% SDS gels
(IRF-3P). The Mtb-induced phosphorylation was rapid and transient,
starting at 1 h and decreasing at 16 h postinfection. The
total IRF-3 content was better evaluated in a shorter gel run (Fig. 2C, lower panel), wherein it appeared to decrease
upon longer infection times, suggesting that proteasome-dependent
degradation could operate in Mtb-infected cells, as previously
described in virus-infected cells (30).
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Type I IFN exerts multiple biological functions through the
activation of STAT-1 and STAT-2 transcription factors. Therefore, the
extent of STAT phosphorylation was analyzed in Mtb-infected DC. A short
kinetics of infection, from 30 min to 8 h (Fig. 3, left panel), and a longer
one up to 48 h (Fig. 3, right panel), were performed.
As a control, immature DC were treated for 30 min with a saturating
dose of IFN-2 (0.1 nM). Weak tyrosine phosphorylation of STAT-1 and
STAT-2 was detected as early as 3 h of infection; it increased at
8 h and started to decrease at 48 h. At any time point, the
extent of phosphorylation was considerably less than in immature DC
treated with exogenous IFN-. Moreover, a significant increase in
STAT-1 and STAT-2 protein levels (3- to 5-fold) was consistently
observed at 16 h (Fig. 3, right panel). This increase
was specific, because the level of the tyrosine kinase 2, involved in
the IFN- signaling pathway, was unchanged (data not shown). To
characterize in more detail the role of Mtb-induced
IFN- production on the observed STAT-1 and STAT-2 activation,
neutralization experiments were conducted. DC cultures were stimulated
with live Mtb in the presence or the absence of neutralizing
anti-IFN Abs (Fig. 3, left panel). A clear
reduction in STAT-1 and STAT-2 phosphorylation was observed in the
presence of anti-IFN- Abs. Moreover, the kinetics of IFN-
production induced by Mtb infection correlated with the kinetics of
STAT activation, strongly suggesting that IFN- mediates the
Mtb-induced STAT phosphorylation.
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Next, we asked whether the Mtb-induced phosphorylation of STAT-1
and STAT-2 correlated with induction of their DNA binding activity.
Among the IFN-stimulated genes induced through STAT activation are
members of IRF family, IRF-1 and IRF-7. In particular, the expression
of IRF-1 is dependent on the activation of STAT-1 recognizing the SBE
motif within the IRF-1 promoter. Thus, binding of STAT-1 homodimers was
analyzed by EMSA on the SBE motif present within the IRF-1 promoter
(52), using whole cell extracts from Mtb-infected DC. A
robust induction of binding activity was evident 8 h after Mtb
infection and remained high at later time points (Fig. 4A). To verify whether this
activity correlated with the induction of the IRF-1 protein, whole cell
extracts were prepared at various times after Mtb infection and were
analyzed for IRF-1 expression by immunoblot. A 1-h infection with Mtb
resulted in induction of the IRF-1 protein, which increased further at
8 h and then leveled off (Fig. 4B). This early rise in
IRF-1 suggests a STAT-independent regulatory mechanism. Because it is
known that IRF-1 induction is also regulated by NF-B (51, 62), we investigated whether the rapid IRF-1 expression observed
in Mtb-infected DC could be ascribed to the activation of the NF-B
pathway. Nuclear extracts were prepared from DC infected with Mtb at
different time points and analyzed by EMSA using the B sequence of
the IRF-1 promoter. As shown in Fig. 4C, Mtb infection
readily activated NF-B DNA binding, which remained high thereafter.
As shown by supershift, the NF-B DNA binding complex contained the
p50 subunit.
In addition, we analyzed the expression of IRF-7 along the infection of
DC because recent studies demonstrated the critical role of this
transcription factor in virus-mediated induction of IFN-
(36, 37, 38). We measured the Mtb-induced STAT DNA binding
activity on the ISRE motif that was recently identified within the
promoter of the IRF-7 gene (53). A low mobility
complex was transiently induced at 8 h postinfection and was
shown, by supershift assays, to contain STAT-2 (Fig. 5A, ISGF-3). A higher mobility
complex was also detected at 8 h after stimulation and persisted
to 48 h. This complex was shown to contain IRF-1 by supershift
assays (Fig. 5A, IRF-1). Consistent with the EMSA
experiment, the IRF-7-specific transcript was detected at 8 h
after Mtb infection and increased at 24 h, as evaluated by
quantitative RT-PCR (Fig. 5B). The IRF-7 protein accumulated
at 16 h after stimulation (Fig. 5C). Altogether, these
strong correlative results suggest that IRF-1 factor and ISGF-3 may
cooperate toward a sustained expression of IRF-7 in Mtb-infected
DC.
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1/13 in Mtb-infected DC
It has been recently described that the relative level of IRF-1,
IRF-3, and IRF-7 modulates the expression profile of IFN-
subtypes (54). To evaluate the relative contribution of
IRF factors to the delayed expression of the IFN-1/13
gene, EMSA experiments were performed on the VRE sequence present
within IFN-1 promoter using whole cell extracts prepared
at various times after Mtb infection (Fig. 6A). Surprisingly, only IRF-1
binding was detected, starting from 8 h after infection. The
identity of the complex was confirmed by supershift experiments using
anti-IRF-1 Abs. As an alternative approach to EMSA, we performed a
DNA affinity purification assay, which represents a more sensitive
analysis of transcription factor binding to target consensus sequences.
Biotinylated oligonucleotides, corresponding to the wild-type VRE of
the IFN-1 promoter and a mutant form, were immobilized onto
streptavidin-coated magnetic beads and then incubated with whole cell
extracts from uninfected or 24-h Mtb-infected DC. The isolated
complexes were examined by immunoblotting with IRF-1-, IRF-3-, and
IRF-7-specific Abs. Despite the presence of both IRF-3 and IRF-7 in the
cell extracts used (data not shown), Mtb infection resulted in
retention of only IRF-1 (Fig. 6B). The binding was specific
because the mutated VRE oligonucleotide did not retain IRF-1 from the
same cell extract.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and consequent induction of cell-mediated immune response. The
importance of CD4+ T cells, IFN- production,
and microbicidal activities in the control of acute
Mycobacterium infections has been confirmed in a variety of
experimental models (47, 48). Moreover, lung human
CD4+ T cells were reported to exert some
cytolytic functions on infected macrophages via Fas ligand-induced
apoptosis (47).
We have previously shown that DC infected with Mtb are primarily
involved in inducing an antimycobacterial T cell immune response
(14). After interacting with the pathogen, DC mature and
acquire the ability to stimulate T cells through surface expression of
MHC and costimulatory molecules, as well as secretion of
immunoregulatory cytokines, such as IL-12 and type I IFN
(14). Given the role played by type I IFN in stimulating
NK cells and CD4+ T lymphocytes, we sought to
characterize the profile of IFN subtypes produced in human DC infected
by Mtb. We show in this work that the contact between Mtb and DC
results in an elevated and rapid expression of IFN-, followed by the
delayed production of IFN-. Whereas in virally infected DC most
IFN- subtypes are induced (E. M. Coccia and G. Uzé,
unpublished observation), in Mtb-infected DC the production is
restricted to the IFN-1/13 subtype, which possesses low antiviral
and antiproliferative activities compared with the others
(63). Whether this IFN subtype exerts some unique
biological effects toward the regulation of immune functions in the
course of Mtb infection is presently unknown.
The kinetics of type I IFN measured in our experimental model is
reminiscent of the two-wave expression profile described in virally
infected fibroblasts, where IFN- production is followed by the
delayed induction of specific IFN- genes (36). The
stimulation of IFN synthesis in virally infected cells is mainly
regulated at the transcriptional level by factors belonging to the
NF-B and the IRF families. While both are important for the
induction of IFN-, NF-B does not participate in IFN-
induction, as no B sequences have been identified in the promoter
region of IFN- genes. It has been reported that Mtb can induce
NF-B activation in human monocytes (64). Accordingly,
we observed the rapid formation of the NF-B complex on the B
sequences of the IFN- VRE, confirming that the
interaction between Mtb and DC readily activates a signaling pathway
leading to NF-B activation. We also investigated the involvement of
IRF family members in the induction of IFN in Mtb-infected DC.
Interestingly, the kinetics of IRF-3 activation paralleled IFN-
induction. Indeed, the rapid IRF-3 phosphorylation, observed within the
first 8 h after infection, is likely involved in the induction of
the IFN- mRNA, whose expression peaks at 16 h following Mtb
infection. Although the involvement of IRF-3 as regulator of IFN gene
expression is extensively documented in studies using recombinant or
ectopically expressed protein (29, 30), EMSA binding of
the endogenous IRF-3 protein to the IFN- promoter has never been
reported, probably reflecting its low affinity to the PRDIII and I
regulatory sequences (33). Accordingly, our attempts in
Mtb-infected DC have been unsuccessful. However, using a chromatin
immunoprecipitation approach, Wathelet et al. (33) were
able to demonstrate virally induced IRF-3 association with the IFN-
promoter. Based on these findings, we propose that IRF-3 and NF-B,
activated by phosphorylation following Mtb infection, might cooperate
to up-regulate the IFN- expression in human DC. Conversely, the
involvement of IRF-3 in the delayed induction of IFN-1/13 is
unlikely, due to its decay at 24 h postinfection.
IFN- secreted from the infected DC is likely to act in an autocrine
and paracrine loop, through the binding to cognate receptors and the
activation of the Janus kinase/STAT pathway. The formation of
heterotrimeric ISGF-3 and homodimeric STAT-1 complexes contributes to
the expression of target genes. Among the genes controlled by the
prompt IFN- production from Mtb-infected DC are IRF-1 and IRF-7,
which could be involved in the second wave of transcription leading to
IFN-1/13 expression. Indeed, the neutralization of IFN- in
Mtb-infected DC abolished IFN- expression (data not shown), and the
expression of IRF-1 and IRF-7 paralleled the IFN-1/13 mRNA
accumulation. Our data suggest that the long-lasting IRF-1 expression
is dependent on STAT-1 homodimers induced by IFN-, whereas the
delayed expression of IRF-7 requires both the IRF-1 and the ISGF-3
complex. IRF-7 was shown to be indispensable for IFN-
gene expression in virally infected human and mouse cells
(36, 37, 38, 65). Despite a consistent increase in IRF-7
content observed upon Mtb infection, our multiple attempts to detect
association of this factor to the IFN-1 VRE sequences by
EMSA or DNA affinity purification assay have failed. One possible
explanation is that the modification(s) induced by Mtb differs, both
quantitatively and/or qualitatively, from those induced by a virus, and
this difference could account for the distinct subsets of IFN-genes induced in the two infectious scenarios. It is conceivable
that IRF-7 undergoes serine phosphorylation following Mtb
infection, because its carboxyl terminus is highly homologous to that
of IRF-3, whose phosphorylation was detected (29, 34). As
recently suggested by Levy et al. (66) in their study of
virus-infected cells, an unknown kinase activated by Mtb infection
could be responsible for IRF-3 and IRF-7 phosphorylation.
Binding of IRF-1 to the PRDIII-I sequences of the IFN-
promoter and to the VRE motif of IFN-1 promoter was
clearly observed starting 8 h after infection. Therefore, IRF-1
could cooperate with IRF-3 and NF-B in the regulation of IFN- and
thereafter with IRF-7 in the regulation of IFN-1/13, arguing for its
repeated involvement in the coordinated expression of type I IFN genes.
Although the original role attributed to IRF-1 in IFN gene induction
was reconsidered following the analysis of IRF-1-null mice (25, 67), IRF-1 was recently shown to direct, together with NF-B
and activating transcription factor-2/c-Jun, the ordered recruitment of
chromatin-modifying and general transcription factors to the
IFN- promoter (68). In addition, it has been
suggested that IRF-1 may directly contribute to the transcriptional
regulation of the IFN-1 and -2 genes
(54). Altogether, these and our results obtained in
Mtb-infected primary cells revamp the role of IRF-1 in the mechanism of
type I IFN gene induction.
Our results also highlight the importance of STAT-1 in the induction of
IRF factors involved in IFN- regulation. Interestingly, the
importance of STAT-1 in the establishment of an antimycobacterial
activity has been demonstrated in patients with unexplained
mycobacterial disease (69). In these subjects, an
impairment of the IFN- response was associated to a defect in STAT-1
activation, due to a heterozygous substitution at nucleotide 2116 that
severely impairs phosphorylation of tyrosine 701 and the subsequent
homodimerization. The formation of a functional ISGF-3 complex in
response to IFN- was observed in these patients. Because both the
IFN-- and the IFN--induced binding of STAT-1 homodimers to SBE
were affected, it would be interesting to measure, in DC obtained from
these patients, IRF-1 expression and IFN- production following Mtb
infection. These studies could help to clarify the role of type I IFN
in the initiation of both innate and adaptive immunity against
Mtb.
The working model that can be drawn from the analysis of the events
occurring in human DC infected in vitro with live Mtb is illustrated in
Fig. 7. The positive feedback regulation
of type I IFN production appears to be mediated by a complex interplay
of NF-B, IRF, and STAT transcription factors, whose coordinated
activation leads to a temporal and stimulus-specific IFN expression. In
our experimental system, we propose IRF-1 as a key factor in the
induction of type I IFN gene expression during Mtb infection.
Interestingly, IRF-1 could be involved as well in a more general
process linked to DC maturation, because it was recently found, by
oligonucleotide microarrays, to be up-regulated in human DC induced to
mature in response to different pathogens (70). The
identification of the gene expression profile associated with DC
maturation in response to Mtb infection will be crucial to the
understanding of the events leading to the development of cell-mediated
immunity and in turn to developing improved therapies to potentiate
protective response against tuberculosis.
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Acknowledgments |
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2b. We are grateful to Schering-Plough
(Innishannon, Ireland) for providing GM-CSF and to Eugenio
Morassi for preparing drawings. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Eliana M. Coccia, Laboratory of Immunology, Istituto Superiore di Sanità, 00161 Rome, Italy. E-mail address: e.coccia{at}iss.it
3 Abbreviations used in this paper: DC, dendritic cell; IRF, IFN regulatory factor; ISGF, IFN-stimulated gene factor; ISRE, IFN-stimulated response element; Mtb, Mycobacterium tuberculosis; PRD, positive regulatory domain; SBE, STAT-binding element; VRE, virus-responsive element.
Received for publication January 16, 2002. Accepted for publication April 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Annu. Rev. Immunol. 15:749.[Medline]
and IFN-: a link between immune memory and chronic inflammation. Immunol. Today 21:337.[Medline]
and as immune regulators: a new look. Immunity 14:661.[Medline]
interferons on immune function. Semin. Oncol. 24:78.
expression. J. Immunol. 165:2028. promoter region. J. Biol. Chem. 268:24032. gene expression. S. L. McKnight, and K. R. Yamamoto, eds. Transcriptional Regulation 1193. Cold Spring Harbor Lab. Press, Cold Spring Harbor, New York.
in virus-induced expression of the IFN- gene. FEBS Lett. 358:225.[Medline]
) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies. Genes Cells 1:995.[Abstract]
genes. FEBS Lett. 425:112.[Medline]
enhancer in vivo. Mol. Cell 1:507.[Medline]
genes by positive feedback through interferon regulatory factor-7. EMBO J. 17:6660.[Medline]
: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157:4781.[Abstract]
activates Stat6 and leads to the formation of Stat2:Stat6 complexes in B cells. J. Immunol. 163:3834. and IFN signalling pathways through synergistic induction of IRF-1/ISGF-2 is mediated by a composite GAS/B promoter element. Nucleic Acids Res. 25:4346. and IFN-, and is likely to autoregulate the p91 gene. EMBO J. 13:158.[Medline]
and 1.9 for the donor
. The ratio of IFN-
and 
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