Cutting Edge: A TLR9 Cytoplasmic Tyrosine Motif Is
Selectively Required for Proinflammatory Cytokine
Annapoorani Chockalingam, William Alfred Rose, II, Maroof Hasan, Chia-Hsin Ju, and
Cynthia Anne Leifer
Compartmentalization of nucleic acid sensing TLR9
has been implicated as a mechanism to prevent recog-
nition of self nucleic acid structures. Furthermore, rec-
ognition of CpG DNA in different endosomal com-
partments leads to the production of the proinflamma-
tory cytokine TNF-a, or type I IFN. We previously
characterized a tyrosine-based motif at aa 888–891 in
the cytoplasmic tail of TLR9 important for appropriate
intracellular localization. In this article, we show that
this motif is selectively required for the production of
TNF, but not IFN. In response to CpG DNA stimu-
lation, the proteolytically processed 80-kDa fragment
is tyrosine phosphorylated. Although Y888 is not itself
phosphorylated, the structure of this motif is necessary
for both TLR9 phosphorylation and TNF-a produc-
tion in response to CpG DNA. We conclude that bi-
furcation in TLR9 signaling is regulated by a critical
tyrosine motif in the cytoplasmic tail.
Immunology, 2012, 188: 527–530.
DNA is an adjuvant for vaccines in nonhuman primates (1).
CpG DNAs are also versatile, because depending on the se-
quence and chemical properties of the CpG DNA, TLR9
signaling can preferentially result in proinflammatory cyto-
kine production and B cell proliferation (CpG DNA-B/D), or
in type I IFN immune production (CpG DNA-A/K) (2, 3).
The outcome of response to the two different classes of CpG
DNAs depends on the endosomal compartment where contact
with TLR9 occurs (4). CpG DNA-A/D and CpG DNA B/K
are endocytosed but then are preferentially retained in early
endosomes (CpG DNA-A) to elicit IFN production or in
lysosomes (CpG DNA-B) to elicit proinflammatory cytokines
(4). TLR9 gains access to these DNAs by trafficking from the
endoplasmic reticulum, through the Golgi (5–7). Recent data
The Journal of
ucleic acid sensing TLRs are important for host de-
fense against pathogens but are also potent immu-
nomodulators. For example, the TLR9 ligand CpG
implicate adaptor protein 3 (AP3) in regulation of TLR9 traf-
ficking from the Golgi to lysosome-related organelles where
IFN production occurs, but whether AP3 is selectively re-
quired for IFN production or is also required for TNF pro-
duction is controversial (8, 9). Regardless, distinct regulatory
mechanisms selectively governing inflammatory cytokine pro-
duction have not been identified.
We hypothesized that one of several discrete TLR9 lo-
calization motifs was phosphorylated and regulated signaling
(YXXF, where X is any amino acid, F is a bulky hydrophobic
amino acid) (10–12). We show that TLR9 with a single point
mutation at Y888 (Y/A) selectively impairs TNF produc-
tion and receptor phosphorylation. Mutation of Y888 to the
structurally conserved phenylalanine (Y/F) retained TNF
production and phosphorylation. We conclude that although
not directly phosphorylated, Y888 was structurally required
for phosphorylation of TLR9, and thereby regulates TLR9-
mediated signal bifurcation.
Materials and Methods
Reagents and plasmids
The following Abs and reagents were used: hemagglutinin (HA; ABM and
Roche), Rab5, phospho-p38 and total p38 (Cell Signaling Technologies),
4G10 (Millipore), CD107a (eBiosciences), secondary Abs (Southern Biotech
and Life Technologies), TNF-a ELISA kit (BioLegend), CpG DNA 2216 and
10104 (Eurofins MWG and Integrated DNA Technologies), LPS (Inviv-
ogen), and DOTAP (Roche). QuikChange (Stratagene) was used for site-
directed mutagenesis of TLR9-HA. Primer sequences are available upon re-
Cell culture, retroviral transduction, and immunoblot analysis
TLR92/2macrophages were cultured in DMEM with 10% (v/v) FBS, 2 mM
L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES (complete DMEM),
and 10 mg/ml ciprofloxacin. CpG DNA–DOTAP complexes were prepared
as previously described (9). Retroviral supernatant with polybrene (8 mg/ml)
was used in spin transductions. Cells were cultured at 37˚C for 44–48 h
before stimulation. Immunoblotting was performed as previously described
RNA (QIAshredder and RNeasy kit; Qiagen) was used to prepare cDNA
(SuperScript III First-Strand Synthesis; Invitrogen). PCR was performed with
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell
University, Ithaca, NY 14853
Received for publication September 22, 2011. Accepted for publication November 18,
This work was supported by National Institutes of Health Grants RO1AI076588 and
RO1AI076588-S1 (to C.A.L.).
A.C., M.H., W.A.R., and C.-H.J. performed experiments; A.C. and C.A.L. designed the
research and wrote the manuscript.
Address correspondence and reprint requests to Dr. Cynthia A. Leifer, Department of
Microbiology and Immunology, College of Veterinary Medicine, VMC C5-153, Cor-
nell University, Ithaca, NY 14853. E-mail address: email@example.com
Abbreviations used in this article: AP3, adaptor protein 3; HA, hemagglutinin; LAMP-1,
lysosomal-associated membrane protein 1; WT, wild-type.
Biosystems). Changes in gene expression were determined using the 22DDCT
TLR92/2macrophages were transduced before treatment with 39 Cy3-10104
CpG DNA, 2216 CpG DNA in a complex with DOTAP or medium. Cells
were stained for HA and CD107a or Rab5 followed by Alexa Fluor 488 and
Alexa Fluor 647 Abs. Coverslips were mounted with Prolong Gold Antifade
Reagent with DAPI (Life Technologies) and visualized on a Leica TCS SP5
confocal microscope using a 633 oil objective (Leica Microsystems). Image
analysis was performed with the colocalization tool. Figures were prepared in
Results and Discussion
A cytoplasmic tyrosine motif is required for TNF production
We previously identified a highly conserved 4-aa motif in the
cytoplasmic tail of TLR9 that contributes to intracellular traf-
ficking (11). To determine whether this motif regulated pro-
duction of the proinflammatory cytokine TNF, IFN, or both,
we retrovirally transduced TLR9-deficient macrophages with
wild-type (WT) TLR9, the tyrosine mutant (TLR9Y888A),
or empty vector, and assayed for TNF-a production in re-
sponse to CpG DNA. Vector-transduced cells did not re-
spond, whereas cells expressing WT TLR9 produced TNF-a
in response to CpG DNA-B (Fig. 1A). However, cells
expressing TLR9Y888Aproduced no TNF-a in response
to CpG DNA-B (Fig. 1A). LPS responses were similar in all
cells (Fig. 1B).
We next asked whether IFN production was also compro-
mised in cells expressing mutant TLR9. WT TLR9, but not
empty vector, reconstituted cells responded toCpG DNA-A in
complex with DOTAP to induce IFN-b mRNA (Fig. 1C).
CpG DNA-A alone, CpG DNA-B alone, or CpG DNA-B in
complex with DOTAP did not induce IFN (Fig. 1C; A.C.
and C.A.L., unpublished observations). However, unlike for
TNF production (Fig. 1A), TLR9Y888Asupported near WT
levels of IFN-b mRNA induction (Fig. 1C). Together, these
data showed that a specific tyrosine motif in the cytoplasmic
tail of TLR9 was selectively required for production of TNF.
TLR9 receptor phosphorylation correlates with TNF production and
depends on Y888
YXXF motifs can be phosphorylated, and thereby regulate
signaling (14); furthermore, TLR9 is phosphorylated in re-
sponse to CpG DNA stimulation (12). Stimulation with CpG
DNA-B resulted in phosphorylation of an 80-kDa form cor-
responding to the mature receptor (Fig. 2A) (13, 15–18).
Full-length TLR9 was not abundant in macrophages, so it is
unclear whether the phosphorylation event occurs exclusively
on p80 or also on full-length TLR9. Regardless, mutation of
Y888 to alanine had no effect on proteolytic processing of
TLR9 (Fig. 2A) but eliminated CpG DNA-dependent p80
phosphorylation (Fig. 2A), suggesting that TLR9 phosphor-
ylation depended on Y888. Mutation of the critical tyrosine
to phenylalanine, a structurally conserved but nonphosphor-
ylated amino acid, did not block phosphorylation. Therefore,
phosphorylation depends on the structural motif, not on
phosphorylation of Y888. Mutation of any one tyrosine in the
proinflammatory cytokine response. A, TLR92/2macrophages were retrovirally
transduced with empty vector (Vector), WT TLR9, TLR9Y888A(Y888A), or
TLR9Y888F(Y888F). Cells were stimulated with media or CpG DNA-B (10
mg/ml) for 8 h. Secreted TNF-a concentration was determined by ELISA.
Error bars indicate SD (n = 4). B, As in A, except cells were stimulated with
LPS (100 ng/ml). C, Vector, WT, or Y888A cells were stimulated with CpG
DNA-A (10 mg/ml) or CpG DNA-B (10 mg/ml) in complex with DOTAP,
and IFN-b mRNA relative expression level (REL%) was determined by real-
time PCR. Results are representative of three experiments.
A tyrosine motif in TLR9 cytoplasmic domain is required for
phosphorylated in response to CpG DNA and support downstream signaling.
TLR92/2macrophages were retrovirally transduced with empty vector (V),
TLR9-HA (WT), TLR9Y888F-HA (Y888F), or TLR9Y888A-HA (Y888A).
Cells were stimulated with CpG DNA-B (10 mg/ml) for the indicated times.
A, HA immunoprecipitates were immunoblotted for phosphotyrosine or HA.
Arrow indicates full-length TLR9; asterisks indicate p80. B, Whole-cell lysates
were immunoblotted for phospho-p38 and total p38. Representative of two
WT TLR9 and TLR9Y888F, but not TLR9Y888A, are tyrosine
528CUTTING EDGE: REGULATION OF TLR9 SIGNALING BY A TYROSINE MOTIF
cytoplasmic tail of TLR9 does not abrogate phosphorylation
(C.A.L., unpublished observations). TLR9 phosphorylation is
complex and likely involves more than one site. WT TLR9,
but not the Y888A mutant, supported downstream signaling
as measured by time-dependent p38 phosphorylation in re-
sponse to CpG DNA-B stimulation (Fig. 2B). Failure of
TLR9Y888Ato support TNF was not due to lack of binding
to ligand, because TLR9Y888Ain lysates interacted with CpG
DNA-B as well as, or better than, WT TLR9 (A.C. and
C.A.L., unpublished observations). Therefore, TLR9 phos-
phorylation occurred selectively on the p80 form of the
receptor, depended on Y888, and correlated with TNF pro-
The structure of Y888 is critical for TNF production
We next asked whether Y888 was directly phosphorylated by
mutation of the critical tyrosine to phenylalanine, which is
structurally related to tyrosine, but cannot be phosphorylated
in TLR92/2macrophages, TLR9Y888Fwas proteolytically
cleaved (Fig. 2A), and supported downstream signaling as
indicated by CpG DNA-induced p38 phosphorylation (Fig.
2B) and TNF-a production in response to CpG DNA-B (Fig.
1A). LPS responses were similar regardless of the transduction
conditions (Fig. 1B). Therefore, although Y888 was not di-
rectly phosphorylated, it was structurally required for TLR9
to support production of TNF.
Y888 is required for TLR9 trafficking to lysosomal-associated
membrane protein 1-positive endolysosomes
We next asked whether the mechanism by which the Y888
motif selectively regulated cytokine production was through
access to endolysosomes. CpG DNA is endocytosed to early
endosomes within 5 min and traffics to late endosomes/
endolysosomes at 1 h (4, 7). The endolysosomal compart-
ment is where proinflammatory cytokine production initiates
and is identified by the presence of lysosomal-associated
membrane protein 1 (LAMP-1). WT and mutant TLR9 were
similarly localized in untreated cells (A.C., W.R., and C.A.L.,
unpublished observations). However, after a 1-h incubation
with 39-Cy3–labeled CpG DNA, WT TLR9 and TLR9Y888F,
but not TLR9Y888A, colocalized with LAMP-1+compart-
ments as determined by four-color confocal microscopy
(Fig. 3). Pearson’s coefficients calculated from line scan
analyses from multiple sections of multiple cells (not shown)
demonstrated colocalization of WT and the F mutant with
CpG DNA (WT: 0.4949, TLR9Y888F: 0.6192) and LAMP-1
(WT: 0.7344, TLR9Y888F: 0.7015; Fig. 3). Pearson’s coloc-
alization coefficients were significantly lower for TLR9Y888A
with CpG DNA (0.1141) and LAMP-1 (0.2186). Together,
these data show that TLR9Y888Afails to induce production of
TNF because it fails to reach LAMP-1+endolysosomes.
Because TLR9Y888Asupported IFN production, we asked
whether TLR9Y888Alocalized with early endosome markers
after stimulation with CpG DNA-A–DOTAP complexes.
Within 10 min of exposure to CpG DNA-A–DOTAP com-
plexes, both WT and TLR9Y888Acolocalized with Rab5 (Fig.
4). Together with the observation that TLR9Y888Adid not
colocalize with LAMP-1 1 h after CpG DNA-B incubation,
TLR9 to the lysosomal compartment after CpG stimulation in macrophages.
Cells were transduced as in Figs. 1 and 2, and stimulated with CpG DNA
10104-Cy3 for 1 h. Overlay of TLR9 (HA, green), CpG DNA (magenta),
lysosomes (CD107a, red), and nuclei (DAPI, blue) is shown to the left. Scale
bars, 5 mm. Representative of 3 experiments, 10 fields per experiment.
Amino acid structure at Y888 is important for trafficking of
early endosomes. Cells were transduced as in Figs. 1 and 2, and stimulated
with CpG DNA 2216 in complex with DOTAP for 10 min. Overlay of
TLR9 (HA, green), early endosomes (Rab5, red), and nuclei (DAPI, blue) is
shown to the left. Scale bars, 5 mm. Representative of 3 experiments, 10 fields
CpG DNA-A induces trafficking of WT and TLR9Y888Ato
The Journal of Immunology 529
we conclude that this structural motif is required to enable Download full-text
trafficking of TLR9 from early endosomes to late endosomes
where proinflammatory signaling is initiated.
TLR9 signals to induce proinflammatory cytokines and IFN
from different endosomal compartments. The mechanisms
regulating this signal bifurcation are not fully understood,
although they likely require AP3 (8, 9). In this study, we
showed that a single tyrosine mutation in the cytoplasmic tail
of TLR9 selectively impairs TNF production. Tyrosine phos-
phorylation of mature TLR9 correlated with proinflamma-
tory cytokine production, but not IFN production, and de-
pended on Y888. Our observation that mutation of Y888 to
alanine inhibits TLR9 phosphorylation, yet selectively impairs
only TNF production, seemingly contradicts observations by
Sanjuan et al. (12). They show that Src kinase inhibitors, such
as PP2, reduce both TNF and IFN production, as well as
inhibit tyrosine phosphorylation of TLR9. However, PP2
inhibition of IFN and TNF responses does not necessarily
mean that blocking tyrosine phosphorylation of TLR9 itself
inhibits both responses. AP3 has recently been implicated in
regulation of TLR9 access to the IFN- and proinflammatory
cytokine-inducing compartments (8, 9). Our motif likely
does bind AP3 because IFN production was normal for
TLR9Y888A. Thus, our data support a model where TLR9
traffics to early endosomes and induces IFN production,
then is sorted to endolysosomal compartments and induces
proinflammatory cytokine production. Together, these motifs,
and the regulatory proteins that bind them, would be
targets to therapeutically manipulate cellular response to CpG
The macrophage cell line derived from TLR9 knockout mice, NR-9569, was
from the National Institutes of Health Biodefense and Emerging Infections
Research Resources Repository, National Institute of Allergy and Infectious
Diseases, National Institutes of Health.
The authors have no financial conflicts of interest.
1. Verthelyi, D. 2006. Adjuvant properties of CpG oligonucleotides in primates.
Methods Mol. Med. 127: 139–158.
2. Verthelyi, D., K. J. Ishii, M. Gursel, F. Takeshita, and D. M. Klinman. 2001.
Human peripheral blood cells differentially recognize and respond to two distinct
CPG motifs. J. Immunol. 166: 2372–2377.
3. Krug, A., S. Rothenfusser, V. Hornung, B. Jahrsdo ¨rfer, S. Blackwell, Z. K. Ballas,
S. Endres, A. M. Krieg, and G. Hartmann. 2001. Identification of CpG oligonu-
cleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic
cells. Eur. J. Immunol. 31: 2154–2163.
4. Honda, K., Y. Ohba, H. Yanai, H. Negishi, T. Mizutani, A. Takaoka, C. Taya, and
T. Taniguchi. 2005. Spatiotemporal regulation of MyD88-IRF-7 signalling for
robust type-I interferon induction. Nature 434: 1035–1040.
5. Chockalingam, A., J. C. Brooks, J. L. Cameron, L. K. Blum, and C. A. Leifer. 2009.
TLR9 traffics through the Golgi complex to localize to endolysosomes and respond
to CpG DNA. Immunol. Cell Biol. 87: 209–217.
6. Leifer, C. A., M. N. Kennedy, A. Mazzoni, C. Lee, M. J. Kruhlak, and D. M. Segal.
2004. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J.
Immunol. 173: 1179–1183.
7. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks,
C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004. TLR9
signals after translocating from the ER to CpG DNA in the lysosome. Nat.
Immunol. 5: 190–198.
8. Blasius, A. L., C. N. Arnold, P. Georgel, S. Rutschmann, Y. Xia, P. Lin, C. Ross,
X. Li, N. G. Smart, and B. Beutler. 2010. Slc15a4, AP-3, and Hermansky-Pudlak
syndrome proteins are required for Toll-like receptor signaling in plasmacytoid
dendritic cells. Proc. Natl. Acad. Sci. USA 107: 19973–19978.
9. Sasai, M., M. M. Linehan, and A. Iwasaki. 2010. Bifurcation of Toll-like receptor
9 signaling by adaptor protein 3. Science 329: 1530–1534.
10. Barton, G. M., J. C. Kagan, and R. Medzhitov. 2006. Intracellular localization of
Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral
DNA. Nat. Immunol. 7: 49–56.
11. Leifer, C. A., J. C. Brooks, K. Hoelzer, J. L. Lopez, M. N. Kennedy, A. Mazzoni,
and D. M. Segal. 2006. Cytoplasmic targeting motifs control localization of toll-like
receptor 9. J. Biol. Chem. 281: 35585–35592.
12. Sanjuan, M. A., N. Rao, K. T. Lai, Y. Gu, S. Sun, A. Fuchs, W. P. Fung-Leung,
M. Colonna, and L. Karlsson. 2006. CpG-induced tyrosine phosphorylation occurs
via a TLR9-independent mechanism and is required for cytokine secretion. J. Cell
Biol. 172: 1057–1068.
13. Chockalingam, A., J. L. Cameron, J. C. Brooks, and C. A. Leifer. 2011. Negative
regulation of signaling by a soluble form of toll-like receptor 9. Eur. J. Immunol. 41:
14. Se ´verin, S., A. Y. Pollitt, L. Navarro-Nun ˜ez, C. A. Nash, D. Moura ˜o-Sa ´, J. A. Eble,
Y. A. Senis, and S. P. Watson. 2011. Syk-dependent phosphorylation of CLEC-2:
a novel mechanism of hem-immunoreceptor tyrosine-based activation motif sig-
naling. J. Biol. Chem. 286: 4107–4116.
15. Ewald, S. E., B. L. Lee, L. Lau, K. E. Wickliffe, G. P. Shi, H. A. Chapman, and
G. M. Barton. 2008. The ectodomain of Toll-like receptor 9 is cleaved to generate
a functional receptor. Nature 456: 658–662.
16. Ewald, S. E., A. Engel, J. Lee, M. Wang, M. Bogyo, and G. M. Barton. 2011.
Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by
cathepsins and asparagine endopeptidase. J. Exp. Med. 208: 643–651.
17. Park, B., M. M. Brinkmann, E. Spooner, C. C. Lee, Y. M. Kim, and H. L. Ploegh.
2008. Proteolytic cleavage in an endolysosomal compartment is required for acti-
vation of Toll-like receptor 9. Nat. Immunol. 9: 1407–1414.
18. Sepulveda, F. E., S. Maschalidi, R. Colisson, L. Heslop, C. Ghirelli, E. Sakka,
A. M. Lennon-Dume ´nil, S. Amigorena, L. Cabanie, and B. Manoury. 2009. Critical
role for asparagine endopeptidase in endocytic Toll-like receptor signaling in den-
dritic cells. Immunity 31: 737–748.
19. Gray, P., A. Dunne, C. Brikos, C. A. Jefferies, S. L. Doyle, and L. A. O’Neill. 2006.
MyD88 adapter-like (Mal) is phosphorylated by Bruton’s tyrosine kinase during
TLR2 and TLR4 signal transduction. J. Biol. Chem. 281: 10489–10495.
530CUTTING EDGE: REGULATION OF TLR9 SIGNALING BY A TYROSINE MOTIF