Competition by inhibitory oligonucleotides prevents
binding of CpG to C-terminal TLR9
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Citation
Avalos, Ana M., and Hidde L. Ploegh. “Competition by inhibitory
oligonucleotides prevents binding of CpG to C-terminal TLR9.”
European Journal of Immunology 41, no. 10 (October 6, 2011):
2820-2827.
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http://dx.doi.org/10.1002/eji.201141563
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Wiley Blackwell
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http://hdl.handle.net/1721.1/84991
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Author Manuscript
Eur J Immunol. Author manuscript; available in PMC 2013 August 19.
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Published in final edited form as:
Eur J Immunol. 2011 October ; 41(10): 2820–2827. doi:10.1002/eji.201141563.
Competition by Inhibitory Oligonucleotides Prevents Binding of
CpG to C-terminal TLR9
Ana M. Avalos and Hidde L. Ploegh
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Nine
Cambridge Center, Cambridge, MA
Summary
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TLR9 recognizes unmethylated CpG-containing DNA commonly found in bacteria. Synthetic
oligonucleotides containing CpG-motifs (CpG ODNs) recapitulate the activation of TLR9 by
microbial DNA, whereas inversion of the CG dinucleotide within the CpG motif to GC (GpC
ODN) renders such ODNs inactive. This difference cannot be attributed to binding of ODNs to the
full-length TLR9 ectodomain, as both CpG and GpC ODNs bind comparably. Activation of
murine TLR9 requires cleavage into an active C-terminal fragment, which binds CpG robustly.
We therefore compared the ability of CpG and GpC ODNs to bind to full-length and C-terminal
TLR9, and their impact on cleavage of TLR9. We found that CpG binds better to C-terminal
TLR9 when compared to GpC, despite comparable and low binding of both ODNs to full-length
TLR9. Neither CpG nor GpC ODN affected TLR9 cleavage in murine RAW 264.7 cells stably
expressing TLR9-Myc. Inhibitory ODNs (IN-ODNs) block TLR9 signaling, but how they do so
remains unclear. We show that IN-ODNs do not impede TLR9 cleavage but bind to C-terminal
TLR9 preferentially, and thereby compete for CpG ODN binding both in RAW cells and TLR9deficient cells transduced with TLR9-Myc. Ligand binding to C-terminal fragment thus
determines the outcome of activation through TLR9.
Keywords
TLR9; CpG ODN; inhibitory ODN; C-terminal TLR9
Introduction
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Toll-Like Receptor 9 (TLR9) is an intracellular DNA sensor specific for unmethylated CpG
motifs, defined as purine-purine-C-G-pyrimidine-pyrimidine [1]. TLR9 recognition of such
sequences, abundantly present in bacterial DNA, triggers a signaling cascade that activates
innate and adaptive immune responses [2]. Synthetic oligonucleotides with CpG motifs
(CpG ODNs) constructed on a phosphorothioate backbone evoke TLR9-dependent
responses and have been instrumental for our current understanding of TLR9 activation and
function in mammalian cells [3].
Stimulatory CpG ODNs have their counterpart in oligonucleotides that are identical in
composition but with an inversion of the CpG sequence, to GpC. CpG and GpC ODNs with
phosphorothioate backbone have similar binding affinities for full-length TLR9 at
physiologically relevant concentrations (0.01-100 nM) [4, 5], therefore the discrimination of
Corresponding author: Hidde L. Ploegh, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, 9
Cambridge Center, Cambridge, MA 02142. Phone 617-324-2031, Fax 617-452-3566. ploegh@wi.mit.edu.
Conflict of interest The authors declare no financial or commercial conflict of interest.
Avalos and Ploegh
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such minor sequence variations is difficult to reconcile with intact receptor-ligand
interactions. In antigen presenting cells of the myeloid lineage, TLR9 resides in the ER and
moves to endolysosomal compartments upon addition of CpG ODN [4, 6]. TLR9 undergoes
proteolytic conversion upon arrival in endolysosomes, a step required for activation [7, 8].
CpG ODNs bind better to cleaved TLR9 than to full length TLR9 [7, 8], suggesting that
processed TLR9 is the physiologically relevant form responsible for sequence recognition
and activation. Expression of the C-terminal fragment of TLR9 is sufficient to endow
TLR9−/− BMDCs with the ability to respond to CpG [8, 9]. Whether C-terminal TLR9 can
distinguish between CpG and GpC sequences is not known.
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In addition to stimulatory and non-stimulatory ODNs, sequences that block responses to
CpG DNA were originally discovered in adenoviruses [10]. Many such inhibitory ODNs
(IN-ODNs), diverse in sequence, inhibit CpG and/or TLR9 activation with varying degrees
of specificity and potency (reviewed in [11]). IN-ODNs were grouped into four classes [11]
based on sequence requirements. Class I IN-ODNs contain the most potent and specific
TLR9 inhibitory ODNs, which block CpG responses in vitro and in vivo [12-16]. These INODNs include a previously identified inhibitory motif characterized by CC(T)
(XXX)3-5GGG [17, 18]. How IN-ODNs decrease TLR9 responses is not known. Neither a
decrease in surface binding nor uptake of CpG [15, 16, 19] can account for inhibition by INODN: both CpG and IN-ODNs are taken up equally well [13, 15]. IN-ODN and CpG bind to
full-length TLR9 with comparable affinities [20] and thus a competitive mechanism of
inhibition, supported by functional data [12, 20] was proposed. Alternatively, binding of INODNs to intact TLR9 could prevent access of proteases to the proteolytic site in the TLR9
molecule, inhibit cleavage and so compromise its activation.
The different outcomes for application of stimulatory, non-stimulatory and inhibitory ODNs
cannot be fully explained by their comparable binding to full-length TLR9 [20]. Since TLR9
requires proteolytic conversion for ligand binding and activation, we hypothesized that ODN
binding to C-terminal TLR9 may be a better correlate for the outcome of the response. Thus,
we evaluated the ability of stimulatory, non-stimulatory and inhibitory ODNs to modulate
TLR9 cleavage, to interact with C-terminal TLR9, and correlated ODN binding with
functional outcomes of TLR9 activation.
Results
GpC ODN fails to efficiently bind C-terminal TLR9
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Upon proteolytic conversion of TLR9, synthetic oligonucleotides containing CpG motifs
(CpG ODNs) bind significantly better to the C-terminal TLR9 fragment when compared to
full length TLR9 [7, 8]. We evaluated the ability of non-stimulatory ODNs containing an
inversion in the CpG motif (GpC ODNs) to bind both intact and C-terminal TLR9, and
compared it to CpG binding. We incubated RAW 264.7 macrophages that stably express
Myc-tagged TLR9 [8] with increasing concentrations of biotinylated CpG (1826 ODN) and
its control GpC ODN. We recovered bound materials on streptavidin (SA)-coated beads and
carried out a digestion with PNGase F to remove complex type N-linked glycans and so
improve resolution by SDS-PAGE. Binding of ODN to full-length or C-terminal TLR9 was
visualized by immunoblot using an anti-Myc antibody. Both bio-CpG and bio-GpC bound to
full-length TLR9 comparably, albeit weakly [4, 5, 20]. However, bio-GpC ODN bound less
avidly to C-terminal TLR9 than did bio-CpG ODN (Figure 1A left panel). Decreased
binding of bio-GpC was not due to less-efficient uptake of this ODN when compared to bioCpG, as both ODNs could be recovered equally with SA beads when analyzed in TBE-urea
acrylamide gels (Supplemental Figure 1). We confirmed that CpG ODN promoted TNFα
production, while GpC ODN failed to do so [15] (Figure 1A right panel). Preferential CpG
ODN binding (and activation) was also observed when ODNs bearing the same sequences as
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CpG and GpC but containing phosphodiester bonds were delivered to RAW-TLR9 Myc
cells using the transfection reagent DOTAP (Supplemental Figure 2). C-terminal TLR9 can
thus distinguish the CG to GC inversion in the sequence of these ODNs whether constructed
on a phosphodiester or a phosphorothioate backbone.
In APCs of myeloid origin, TLR9 resides in the ER, and trafficks to endolysosomal
compartments upon addition of CpG [4, 6]. In these compartments, TLR9 cleavage occurs
[7, 8]. In unstimulated Myc-tagged TLR9-expressing RAW macrophages, a sizable fraction
of TLR9 appears to traffic to endolysosomes, since the C-terminal fragment of TLR9 is
readily detected in pulse-chase experiments also in the absence of CpG [8]. We
hypothesized that addition of CpG to RAW macrophages would enhance cleavage of TLR9
by stimulating the movement of full length TLR9 to lysosomal compartments, whereas
addition of GpC would not affect cleavage. We therefore performed pulse-chase
experiments where RAW cells expressing Myc-tagged TLR9 were pulsed for 2 h and chased
for 0, 2 or 5 hours, in the absence or presence of CpG or GpC. TLR9 was recovered by
immunoprecipitation with anti-Myc antibodies, and digested with PNGase F. We observed
no differences in cleavage of TLR9 in cells treated with CpG compared to untreated controls
(Figure 1B). At rest, a significant pool of C-terminal TLR9 must already be available for
activation by ODNs, and addition of CpG ODN does not enhance trafficking of TLR9 in
these cells.
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Inhibitory ODNs do not affect TLR9 cleavage
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DNA sequences with CpG inhibitory activity (IN-ODNs) were originally identified in
adenoviral serotypes as ODNs capable of suppressing responses to E.coli DNA [10]. The
mechanism by which IN-ODNs block TLR9 responses has not been elucidated. Since TLR9
requires proteolytic cleavage for activation, we asked whether inhibitory ODNs prevented
TLR9 activation by affecting its proteolytic conversion. To test this possibility, we used the
Class I inhibitory sequences 2088 and G-ODN. ODN 2088 blocks CpG-dependent IL-6
production, apoptosis and proliferation in B cells [16, 21], is protective in vivo in a mouse
model of diabetes [22] and counteracts the acute inflammatory response triggered by
adenoviral infection [23]. The IN-ODN G-ODN blocks CpG responses in murine RAW
macrophages and BMDC in vitro, and in human pDCs. G-ODN is also protective in a mouse
model of cytokine-mediated lethal shock [15]. Both of these inhibitory ODNs contain a
sequence element deemed the inhibitory motif, characterized by a CC(T) sequence, followed
by three to five residues and the G triad GGG [17, 18]. To test whether 2088 and G-ODN
blocked TLR9 cleavage, we performed pulse-chase experiments on Myc-TLR9 expressingRAW macrophages in the presence of these IN-ODN, or in the presence of the cysteine
protease inhibitor z-FA-FMK [24]. Treatment with IN-ODN did not change the cleavage
profile of untreated cells, unlike treatment with z-FA-FMK, which blocks TLR9 cleavage
(Figure 2). The apparent molecular weight of the C-terminal fragment was indistinguishable
for all conditions examined and thus the site of cleavage is unlikely to be affected by
inclusion of the different ODNs. IN-ODN 2088 on occasion appeared to promote cleavage
at a slightly higher rate than seen for G-ODN or in untreated cells, but this was not observed
consistently (Figure 2B). We conclude that inhibitory ODNs affect neither the rate of TLR9
cleavage nor the identity of the cleavage products generated.
IN-ODNs bind C-terminal TLR9 preferentially
Inhibitory ODN (IN-ODN) may block CpG responses through a competitive mechanism
[12, 20]. We hypothesized that IN-ODNs may bind to C-terminal TLR9 without leading to
activation, and thus not only prevent signal transduction but also binding of stimulatory CpG
ODNs. Since IN-ODNs interact with intact TLR9, we compared binding of increasing
concentrations of biotinylated versions of the IN-ODNs 2088 and G-ODN to both full-
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length and C-terminal TLR9. Both ODNs showed significant binding to C-terminal TLR9
(Figure 3A). To compare binding of CpG, GpC and IN-ODNs to C-terminal TLR9, we
incubated RAW macrophages with 1 μM each of biotinylated CpG, GpC and IN-ODNs.
Inhibitory ODNs bind to C-terminal TLR9 better than CpG ODN (Figure 3B). A
biotinylated version of the TLR4 ligand LPS failed to bind TLR9 in either full-length or its
cleaved form (Figure 3B). Comparable amounts of biotinylated CpG, GpC and 2088 ODNs
were found inside cells by SA-precipitations and detection in TBE-Urea Acrylamide gels.
However G-ODN was found in higher concentrations inside cells (Supplemental Figure 1).
Binding of IN-ODN to C-terminal TLR9 may therefore block activation simply through
competitive binding to cleaved TLR9.
IN-ODNs block binding of CpG to C-terminal TLR9
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To test whether IN-ODN may block TLR9 activation through competitive binding, we
incubated TLR9-Myc-expressing RAW macrophages with increasing concentrations of
unlabelled 2088 or G-ODN for 15 min, and then added 1 μM biotinylated CpG ODN for 2
h. Binding to C-terminal TLR9 was detected by recovery on streptavidin beads followed by
immunoblotting using an anti-Myc antibody. Addition of G-ODN or 2088 inhibited binding
of biotinylated CpG to C-terminal TLR9 (Figure 4A top two panels) in a manner that
correlated with their ability to block TLR9-dependent TNFα production in response to CpG
ODN (Figure 4B, top two panels). As expected, based on the low level of binding of GpC
ODN to C-terminal TLR9, addition of GpC ODN did not affect binding of biotinylated CpG
ODN to C-terminal TLR9 (Figure 4A bottom panel), but modestly blocked CpG ODN
responses at high concentrations (Figure 4B bottom panel and [15]).
To eliminate the potential confounding effect of endogenous TLR9 in binding of BIO-CpG
to TLR9, we transduced bone marrow-derived macrophages (BMDM) from TLR9−/− mice
with retrovirus expressing Myc-tagged TLR9. TLR9-deficient cells transduced with TLR9Myc responded to CpG ODN, and this activation was blocked by addition of IN-ODN 2088
(Figure 5A). As expected, addition of non-stimulatory GpC ODN did not affect the cytokine
response of TLR9−/− BMDM transduced with TLR9-Myc. BIO-CpG bound preferentially
to C-terminal TLR9, and this binding was prevented by addition of unlabelled 2088 but not
by addition of GpC ODN (Figure 5B). We conclude that IN-ODN block activation of TLR9
responses through competitive binding to C-terminal TLR9.
Discussion
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Activation of TLR9 requires two steps: ligand binding to the receptor and initiation of
signaling by recruitment of MyD88 to the TIR domains present in the cytoplasmic region of
TLR9 [25]. The presence of CpG motifs or CG repeats in both synthetic and natural DNA
sequences is required for TLR9 signaling [1, 26]. So far it has been not possible to correlate
TLR9 function with ODN binding to the intact TLR9 ectodomain. We propose that binding
to C-terminal TLR9 and not to full-length TLR9 is the first step toward activation of TLR9.
GpC ODN fails to activate TLR9 because it is unable to bind cleaved TLR9. In contrast, INODNs bind C-terminal TLR9 but are unable to trigger responses due to the lack of CpG
motifs. CpG ODNs both bind and include immunostimulatory CpG sequences and therefore
induce activation. It is noteworthy that C-terminal TLR9 discriminates between the
orientation of the CG repeat in the CpG motif, considering the overall similar properties of
CpG and GpC ODNs. This result underscores the importance of cleavage for fine-tuning of
sequence-specific recognition of ODNs by TLR9.
Perhaps unexpectedly, CpG ODN did not enhance cleavage of TLR9 in RAW cells even
though translocation of TLR9 to lysosomes in human pDCs and mouse macrophages has
been shown to be CpG dependent [4]. The pool of cleaved TLR9 present in the
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endolysosomes in resting macrophages appears to be sufficient for CpG to promote
activation, as this ODN reaches these compartments within 30 min of stimulation [27]. In
this regard, RAW macrophages behave very much like primary murine immune cells of
myeloid and lymphoid origin, where a significant amount of cleaved TLR9 is already found
at rest (Brinkmann, Avalos, Ploegh et al, manuscript in preparation).
GpC ODNs bind only weakly to C-terminal TLR9, which accounts for their inability to fully
block CpG binding to C-terminal TLR9, or to significantly decrease activation by CpG
ODN at suboptimal and physiologically relevant concentrations (0.1-1 μM), in spite of the
reported potential inhibitory effects exerted by these GpC at high concentrations [4, 20].
Binding of IN-ODNs to full-length TLR9 in all cases was very weak or undetectable,
(Figure 3, and results not shown), even though the lysates used for immunoprecipitation
contained comparable amounts of full-length and C-terminal TLR9 (Figure 1A and 3, lower
panels). As shown for CpG ODNs, IN-ODNs may rapidly traffick to endolysosomes and
bind preferentially to the existing pool of C-terminal TLR9, leaving little available ODN to
bind the remaining intact TLR9 in these compartments. Consistent with this idea and with
the results shown above, Class I IN-ODNs cause a decrease in colocalization of TLR9 and
CpG in vesicles of possible endolysosomal origin [13].
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Binding alone to C-terminal TLR9 is not sufficient for activation. In the case of IN-ODN
2088 and G-ODN, binding to C-terminal TLR9 did not translate into signaling and
activation, possibly due to the absence of a canonical CpG motif, or a significant number of
CG repeats in these ODNs. The identification of the minimal sequence required for binding
to C-terminal TLR9 was beyond the scope of this manuscript, but preferential binding of INODNs to C-terminal TLR9 may be facilitated by the formation of G-tetrads between ODN
molecules [28], leading to larger structures able to obstruct CpG binding due to steric
hindrance. Thus, the presence of the inhibitory motif CC(T)(XXX)3-5GGG may allow INODN binding, and an increased number of G residues may favor interaction and/or uptake.
The direct binding results shown here eliminate possible non-specific inhibitory effects due
to interactions with other receptors for G-rich ODNs, such as scavenger receptors, as has
been shown for other G-rich inhibitory ODNs [18]. Thus, the direct correlation between
competition for binding and inhibition of cytokine production shown here, as well as the
direct binding data, further clarify the action of at least 2088 and G-ODN on TLR9. The
specificity of action of 2088 in spite of the possible formation of G-tetrads is corroborated
by the finding that a deaza modification of a single G in ODN 2088, which should prevent
G-tetrad formation, did not affect its inhibitory activity [16].
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The deoxyribose backbone is claimed to be responsible for activation and binding of ODN
to intact TLR9 irrespective of sequence [29]. However, sequence-dependent TLR9
activation of B cells by natural DNA sequences has also been recorded [26], and differences
in concentration and uptake mechanisms may account for these discrepancies. Macrophages
are less efficient at taking up phosphodiester backbone ODNs (PO-ODNs) when compared
to their phosphorothioate couterparts [30] and therefore this system is not typically
amenable for binding studies with PO-ODNs. To overcome this handicap, we delivered CpG
and GpC ODNs containing phosphodiester bond using the transfection reagent DOTAP, a
reagent used before to deliver TLR9 ligands to the endolysosome [29]. We and found that
still C-terminal TLR9 could discriminate between CpG and GpC motifs independently of
the nature of the backbone, clearly demonstrating that C-terminal TLR9 can distinguish
these subtle sequence differences present in these ODNs.
Interestingly, mutations in N-terminal domain of TLR9 rendered the receptor inactive,
suggesting that the N-terminal domain may exert some regulatory role on the activity of
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TLR9 [31], perhaps by controlling the folding pathway of the nascent TLR9 polypeptide.
The definitive answer as to how synthetic oligonucleotides and natural DNA sequences bind
and induce conformational changes that lead to TLR9 signaling can be obtained only upon
resolution of the structure of the TLR9 fragments resulting from ectodomain cleavage in the
absence or presence of its ligands. The results shown here underscore the importance of
studying the physical properties of C-terminal TLR9 as well as intact TLR9 and their
agonists and antagonists to better understand the function of this receptor.
Materials and Methods
Cells and Reagents
RAW 264.7 macrophages stably expressing TLR9 with a Myc tag have been previously
described [8]. Phosphothioate and phosphodiester backbone oligonucleotides 1826 (CpG)
(5′-TCCATGACGTTCCTGACGTT-3′) and 1826 control (GpC) (5′TCCATGAGCTTCCTGAGCTT-3′), and phosphothioate ODNs 2088 (5′TCCTGGCGGGGAAGT-3′), G-ODN (5′-CTCCTATTGGGGGTTTCCTAT-3′) and their
biotinylated counterparts were from Integrated DNA Technologies (IDT). z-FA-FMK and
LPS were from Sigma. Biotinylated LPS was from Invivogen.
Pulse chase analysis
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RAW macrophages were starved in DME medium lacking cysteine and methionine and
without serum for 1h. Cells were then incubated with DME lacking cysteine and methionine
in the presence [35S] methionine and [35S] cysteine (Perkin Elmer) at 0.2 mCi/ml and
dialyzed serum for 2 hours (“pulse”). Cells were washed three times with 10% FBS normal
DME medium and incubated (“chased”) for the indicated times. During all three periods
(starvation, pulse and chase) cells were incubated with ODNs or z-FA-FMK as indicated in
the text. Cells were then lysed in 1% digitonin, radioactive labeling quantified using a
scintillation counter, and Myc-TLR9 was immunoprecipitated and reimmunoprecipitated (IP
and reIP) using anti-Myc antibody and protein G beads in lysates bearing equal cpm. Eluates
from Protein G beads were digested using PGNase F and protein separated by SDS-PAGE
and visualized in autoradiogram. Densitometry was performed by exposing gels to
phosphorimager plates and analyzing the intensity of bands using the BAS-2500
Phosphoimager and Multi Gauge software (Fujifilm).
Binding of biotinylated ODNs to TLR9
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TLR9-Myc expressing RAW macrophages or BMDM from TLR9−/− mice transduced with
TLR9-Myc were stimulated for 2 h with biotinylated oligonucleotides and washed once with
PBS. For competition studies, TLR9-Myc expressing RAW macrophages or BMDM from
TLR9−/− mice transduced with TLR9-Myc were stimulated with unlabelled 2088, G-ODN
or GpC for 15 min, then biotinylated CpG was added and stimulated for another 2 h. Plates
were washed once with PBS before lysis. Cells were lysed using 1% digitonin buffer
containing protease inhibitors. Biotin-ODN/protein conjugates were precipitated from
lysates containing equal protein concentrations using streptavidin (SA) beads and digested
with PGNase F. Immunoprecipitates and total lysates were run in SDS-PAGE,
immunoblotted onto PVDF membranes and developed using anti-Myc antibody.
Retroviral transduction
HEK293T cells were transfected with plasmids encoding TLR9-Myc, VSV-G and Gag-Pol
proteins. After 48 hours, supernatants containing virus were collected and filtered through a
0.45 μm filter. Virus-containing supernatants were added to bone marrow cells from
TLR9−/− mice one day after they were harvested from the bone and incubated for 24 h in
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the presence of 5% M-CSF. On the next day, cells were given fresh media in 5% M-CSF
and cultured for 5 days. On day 6 cells were assayed for TLR9 function and ODN binding.
The viral transduction efficiency was ~15%.
TNFα production assays
For detection of secreted TNFα, 0.5×106 cells were seeded in 96-well plates, stimulated for
2 h and supernatants harvested. Titers of TNFα were determined using the OptEIA TNFα
ELISA kit (Becton Dickinson) according to manufacturers specifications. For detection of
intracellular TNFα, BMDM from TLR9−/− mice transduced with TLR9-Myc retrovirus
were stimulated for 2 h in the presence of Brefeldin A. Cells were then trypsinized,
permeabilized with 0.5% saponin in 5% FBS/PBS buffer, incubated with Alexa647-labelled
anti-TNFα antibody (BD Biosciences) for 30 min and analyzed in LSRII flow cytometer
(Becton Dickinson). MFI were calculated using the FlowJo software (Tree Star).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
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The authors wish to thank Boyoun Park for scientific discussion and technical support. This work was supported
with grants from NIH to H.L.P. A.M.A. is a recipient of the Merck-MIT Postdoctoral Fellowship.
Abbreviations
ODN
Oligonucleotide
TLR9
Toll-Like Receptor 9
MyD88
Myeloid Differentiation Primary Response Gene 88
TIR
Toll/Interleukin-1 Receptor
z-FA-FMK
Benzyloxycarbonyl-Phenyl-Alanyl-Fluoromethylketone
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Figure 1. CpG ODN bind better to C-terminal TLR9 when compared to GpC ODN
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(A) (Left) Top panel: RAW macrophages expressing TLR9-Myc were incubated with
increasing concentrations of biotinylated CpG or GpC or left untreated (−) for 2 h. CpGbound materials were recovered using streptavidin beads and analyzed by immunoblotting
with anti-Myc antibody. Bottom panel: TLR9-Myc levels were detected in total lysates by
immunoblotting with anti-Myc antibody. (Right) RAW macrophages expressing TLR9-Myc
were stimulated with increasing concentrations of CpG and GpC for 2 h, supernatants
harvested and TNFα production measured by ELISA. (B) TLR9-Myc expressing RAW
macrophages incubated with 1 μM CpG or GpC or left untreated (no addition) were pulsed
for 2 h with [35S] cysteine/methionine and chased for 0, 2 and 5 h. Proteins were
immunoprecipitated and reimmunoprecipitated using anti-Myc antibody, and analyzed by
SDS-PAGE and autoradiography. Individual band intensity was analyzed by
phosphorimaging, and shown are the intensity ratios of C-terminal (C-term)/intact TLR9. In
A and B, noted are the locations of full length (intact) and C-terminal (C-term) TLR9.
Results in (A) are representative of six and two independent experiments for left and right
panels respectively. In (B), results shown are representative (left) and the mean±SD (right)
of two independent experiments.
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Figure 2. IN-ODN do not affect TLR9 cleavage
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(A) RAW macrophages expressing TLR9-Myc were incubated with 1 μM of the IN-ODN
2088 and G-ODN, 20 μM of the cysteine protease inhibitor z-FA-FMK, or left untreated (no
addition) during 1 h starvation, 2 h pulse with [35S] cysteine/methionine and 0, 2 and 5 h
chase. Proteins were immunoprecipitated and reimmunoprecipitated using anti-Myc
antibody, and analyzed by SDS-PAGE and autoradiography. Noted are the locations of full
length (intact) and C-terminal (C-term) TLR9. (B) Individual band intensity was analyzed
by phosphorimaging, and shown are the intensity ratios of C-terminal (C-term)/intact TLR9.
Results shown are representative (A) and the mean±SEM (B) of three independent
experiments.
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Figure 3. IN-ODN bind to C-terminal TLR9
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(A) RAW macrophages expressing TLR9-Myc were incubated with increasing
concentrations of biotinylated G-ODN (left, 0.1, 0.25 and 0.5 μM) or 2088 (right, 0.1, 0.25
and 1 μM) for 2 h or left untreated (−). Biotin ODN/protein conjugates were recovered with
streptavidin beads and analyzed by immunoblotting using anti-Myc. (B) RAW macrophages
expressing TLR9-Myc were incubated with 1 μM biotinylated CpG, GpC, 2088, G-ODN
(G-O), 100 ng/ml of biotinylated LPS or left untreated (−). Precipitation was performed
using SA beads and bound materials analyzed by immunoblotting using anti-Myc. In A and
B, bottom panels show TLR9-Myc levels in total lysates. Noted are the locations of full
length (intact) and C-terminal (C-term) TLR9. Shown are representative results of three
independent experiments.
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Figure 4. IN-ODNs block binding of CpG ODN to C-terminal TLR9
(A) RAW macrophages expressing TLR9-Myc were incubated for 15 min with increasing
concentrations of unlabeled 2088 (top), G-ODN (middle) and GpC (bottom) ODN before
addition of 1 μM biotinylated CpG (A, *CpG) or media (−) and incubated for 2h. CpGbound materials were recovered using streptavidin beads and analyzed by immunoblotting
using anti-Myc antibody. (B) Cells pretreated under the same conditions as in (A) were
incubated with 0.1 μM CpG or 20 ng/ml LPS for 2 h. Cell supernatants were analyzed for
production of TNFα by ELISA. In (A), noted are the locations of full length (intact) and Cterminal (C-term) TLR9. Shown are representative experiments of five (2088), four (GODN) and two (GpC) in (A), and two (2088), four (G-ODN) and two (GpC) in (B).
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Figure 5. IN-ODNs prevent binding and activation of TLR9 in BMDM from TLR9−/−
transduced with TLR9-Myc
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Bone marrow cells from TLR9−/− mice were transduced with retrovirus expressing TLR9Myc. At day 5 post-transduction, in (A) TLR9−/− BMDM transduced with TLR9-Myc
(white bars) or WT BMDM (black bars) were unstimulated or preincubated with 1μM 2088
or GpC for 15 min, and then stimulated with 1μM of CpG or with 0.1 μM R848 for 4 h in
the presence of 10μg/mL Brefeldin A. Intracellular TNFα was measured by flow cytometry
and median fluroscence intensity (MFI) calculated. Data shown is the mean+SD MFI of two
independent experiments after substracting MFI obtained in the unstimulated condition (222
for TLR9−/− and 262 for WT). In (B), TLR9−/− BMDM were either not transduced (−) or
transduced with TLR9-Myc. Transduced cells were unstimulated (“no CpG”), or
preincubated with media (−), 0.5 μM 2088 or GpC for 15 min before addition of 0.5 μM
BIO-CpG and incubated for 2 h. Cells were lysed and precipitation with SA-beads was
performed. SA-recovered materials were analyzed by immunoblot using anti-Myc antibody.
Shown is a representative result from two independent experiments.
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