Articles
Constitutive MHC class I molecules negatively
regulate TLR-triggered inflammatory responses via
the Fps–SHP-2 pathway
npg
© 2012 Nature America, Inc. All rights reserved.
Sheng Xu1,4, Xingguang Liu1,4, Yan Bao1, Xuhui Zhu1, Chaofeng Han1, Peng Zhang1, Xuemin Zhang2,
Weihua Li2 & Xuetao Cao1,3
The molecular mechanisms that fine-tune Toll-like receptor (TLR)-triggered innate inflammatory responses remain to be fully
elucidated. Major histocompatibility complex (MHC) molecules can mediate reverse signaling and have nonclassical functions.
Here we found that constitutively expressed membrane MHC class I molecules attenuated TLR-triggered innate inflammatory
responses via reverse signaling, which protected mice from sepsis. The intracellular domain of MHC class I molecules was
phosphorylated by the kinase Src after TLR activation, then the tyrosine kinase Fps was recruited via its Src homology 2 domain
to phosphorylated MHC class I molecules. This led to enhanced Fps activity and recruitment of the phosphatase SHP-2, which
interfered with TLR signaling mediated by the signaling molecule TRAF6. Thus, constitutive MHC class I molecules engage in
crosstalk with TLR signaling via the Fps–SHP-2 pathway and control TLR-triggered innate inflammatory responses.
Toll-like receptors (TLRs) are key pattern-recognition receptors
used by cells of the innate immune system to detect the conserved
­components of pathogens, and they have critical roles in host defense
against invading microbial pathogens1,2. After the recognition of
pathogen-associated molecule patterns, TLRs initiate innate immune
responses by activating signaling pathways that depend on the adaptor
MyD88 or the adaptor TRIF and consequently induce the production of proinflammatory cytokines and type I interferon 3. Many
factors have been identified as being essential for full activation of
TLR responses; however, inhibitors of TLR pathways need further
investigation, as inappropriate activation or overactivation of TLR
signaling may result in inflammatory disorders such as septic shock
or autoimmune diseases4. The identification of the negative regulators
and details of the mechanisms by which TLR signaling is fine tuned
remain to be fully elucidated.
Major histocompatibility complex (MHC) class I molecules are
expressed on all nucleated cells from vertebrates. Classical MHC
class I molecules (HLA-I in humans; H-2 I in mice) are heterodimers
composed of β2-microglobulin (β2m) and a membrane-bound heavy
chain with three extracellular domains and a cytoplasmic domain of
about 40 amino acids5. The component β2m is essential for the stable
expression of MHC class I, and β2m-deficient mice lack most if not all
MHC class I molecules6. The main function of MHC class I molecules
seems to be the presentation on the cell surface of small-peptide fragments of endogenously produced antigens for recognition by CD8+
cytotoxic T lymphocytes6. MHC class I molecules also contribute to the
development and selection of CD8+ T cells in the thymus7, as well as
to the ‘education’ and tolerance of natural killer (NK) cells8,9. Normal
expression of MHC class I molecules by target cells inhibits the lysis of
target cells by NK cells. However, chronic interaction between MHC
class I molecules and cognate inhibitory receptors is essential for the
licensing of NK cells and for the acquisition of their killing ability9,10.
In these cases, MHC class I molecules act as ligands to stimulate
relevant receptors to trigger downstream signaling.
In addition to the classical function of antigen presentation, MHC
class I molecules can also mediate reverse signaling after ligation
and have nonclassical functions11–13. The aggregation of MHC class
I molecules on the cell surface with agonist antibodies, T cell antigen
receptors (TCRs) or the coreceptor CD8 activates signaling pathways
in T cells, B cells, tumor cells or endothelial cells and elicits ­various
biological effects, such as cell apoptosis, activation or proliferation13–15. Crosslinkage of MHC class I molecules on human NK cells
induces intracellular tyrosine phosphorylation and inhibits NK cell
cytotoxicity directed against tumor target cells16. Such data suggest
that MHC class I molecules can be ligands and signaling receptors
themselves to mediate reverse signaling via association with other
receptors or directly through aggregation. The activation of tyrosine
kinases, including Lck, Lyn, Syk, Zap70 and Tyk2, occurs immediately after engagement of MHC class I molecules 17,18. So far, almost
all data about the nonclassical functions of MHC class I molecules
have been obtained from studies of lymphoid cells; however, whether
myeloid cells or antigen-presenting cells (APCs) display MHC class I
molecules with nonclassical functions and reverse signaling has not
been elucidated. Intracellular MHC class II molecules do promote
1National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China. 2National Center of Biomedical
Analysis, Beijing, China. 3National Key Laboratory of Molecular Biology, Chinese Academy of Medical Sciences, Beijing, China. 4These authors contributed equally to
this work. Correspondence should be addressed to X.C. (caoxt@immunol.org).
Received 28 October 2011; accepted 6 March 2012; published online 22 April 2012; corrected online 4 May 2012; doi:10.1038/ni.2283
nature immunology VOLUME 13 NUMBER 6 JUNE 2012
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Articles
npg
© 2012 Nature America, Inc. All rights reserved.
RESULTS
MHC class I deficiency exacerbates TLR-triggered responses
To investigate the role of MHC class I molecules in TLR-triggered
innate inflammatory responses, we challenged β2-­microglobulindeficient (B2m−/−) mice with various TLR ligands, including
lipopolysaccharide (LPS), the synthetic RNA duplex poly(I:C) or an
oligodeoxynucleotide based on the dinucleotide motif CpG (CpG
ODN). MHC class I–deficient mice produced significantly more
tumor necrosis factor (TNF), interleukin 6 (IL-6) and interferon-β
(IFN-β) than did their littermates (control mice; Fig. 1a). We detected
no substantial difference between B2m−/− and wild-type mice in the
development of myeloid cells or the expression of TLRs (data not
shown), which excluded the possibility that the phenomenon noted
above was due to the abnormal development of myeloid cells or TLR
expression. Furthermore, after lethal challenge with LPS, almost all
MHC class I–deficient mice died within 24 h, whereas 40% of their littermates (control mice) were alive at that time and about 25% of these
survived the challenge (Fig. 1b). Consistent with that, we observed
more severe infiltration of polymorphonuclear cells and interstitial
pneumonitis in the lungs of MHC class I–deficient mice 8 h after
LPS challenge (Fig. 1c). Furthermore, we also confirmed the greater
production of TNF, IL-6 and IFN-β in mice deficient in the MHC
class I heavy chain (H2-K1−/−H2-D1−/−; called ‘KbDb−/−’ here) after
TLR challenge (Supplementary Fig. 1a).
Given that MHC class I–deficient mice have considerably fewer
CD8+ T cells in the thymus, spleen and lymph nodes, we further reconstituted lethally irradiated wild-type mice with wild-type, B2m−/− or
552
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IL-6 (ng/ml)
TLR-triggered innate immune responses by maintaining activation
of the kinase Btk19. Whether and how MHC class I molecules intersect with innate TLR signaling pathways and regulate TLR-triggered
innate inflammatory response remains unknown.
Here we demonstrate that deficiency in MHC class I resulted in
enhanced TLR-triggered production of proinflammatory cytokines
and type I interferon both in vitro and in vivo. Consistent with that,
engagement of MHC class I molecules inhibited TLR-triggered signal
transduction and production of cytokines. Constitutive membrane
MHC class I molecules physiologically interacted with the tyrosine
kinase Fps (Fes). After the stimulation of TLRs with ligands, MHC
class I molecules became phosphorylated and recruited more Fps,
which resulted in more potent activation of the phosphatase SHP-2
and, finally, suppressed TLR-triggered inflammatory responses.
Therefore, our results demonstrate a nonclassical function for MHC
class I molecules as negative regulators of a TLR pathway, which adds
new insight into the fine tuning of TLR-triggered innate inflammatory responses.
a
TNF (ng/ml)
Figure 1 MHC class I–deficient mice are more susceptible to TLR
challenge. (a) Enzyme-linked immunosorbent assay (ELISA) of TNF,
IL-6 and IFN-β in serum from B2m+/+ and B2m−/− mice (n = 5 per
genotype) 2 h after intraperitoneal challenge with PBS, LPS, poly(I:C)
or CpG-ODN. (b) Survival of B2m+/+ and B2m−/− mice (n = 10 per
genotype) after lethal challenge with LPS (10 mg per kg body weight).
P < 0.01 (Wilcoxon test). (c) Hematoxylin-and-eosin staining of lungs from
B2m+/+ and B2m−/− mice 8 h after challenge with PBS or LPS. Original
magnification, ×100. (d) ELISA of TNF, IL-6 and IFN-β in serum from
wild-type mice (n = 6 per group) reconstituted for 8 weeks with B2m+/+
bone marrow cells (B2m+/+→B2m+/+) or B2m−/− bone marrow cells
(B2m−/−→B2m+/+), followed by challenge with PBS or LPS and analysis
2 h later. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from
three independent experiments (a,d; mean ± s.e.m.) or are representative
of three independent experiments with similar results (b,c).
2.0
**
LPS
B2m+/+→B2m+/+
B2m–/–→B2m+/+
1.5
1.0
0.5
0
PBS LPS
KbDb−/− bone marrow cells and then assessed their responses to challenge with the TLR ligands. When challenged with LPS, chimeric
mice reconstituted with B2m−/− or KbDb−/− bone marrow produced
more TNF, IL-6 and IFN-β than did chimeric mice reconstituted with
wild-type bone marrow (Fig. 1d and data not shown). Thus, these
data demonstrated that TLR-triggered inflammatory responses were
greater in MHC class I–deficient mice, which indicated a suppressive
role for MHC class I molecules in the TLR response.
To investigate the role of MHC class I molecules in host resistance to pathogen infection, we challenged B2m−/− mice and KbDb−/−
mice with Gram-negative Escherichia coli or Gram-positive Listeria
­monocytogenes. After infection with E. coli, the production of TNF,
IL-6 and IFN-β in MHC class I–deficient mice was significantly
greater than that of their littermates (control mice; Fig. 2a and
Supplementary Fig. 1b). Accordingly, MHC class I–deficient
mice had a greater load of E. coli bacteria in the blood (Fig. 2b and
Supplementary Fig. 1b), consistent with published findings that
proinflammatory cytokines promote the dissemination of E. coli20.
After infection with L. monocytogenes, MHC class I–deficient mice
also produced more proinflammatory cytokines and had a smaller
bacterial load in the spleen and liver (Fig. 2c,d and Supplementary
Fig. 1c). Furthermore, consistent with published reports21, NK cells
from MHC class I–deficient mice showed less cytotoxicity directed
against target cells and produced less IFN-γ after infection with
L. monocytogenes (data not shown), which excluded the possibility
that the smaller bacterial load in MHC class I–deficient mice was a
result of enhanced killing by NK cells. These data indicated that MHC
class I molecules may help the host restrict inflammatory responses
after bacterial infection and protect the host from inflammatory injuries mediated by innate immune responses.
More cytokine production in MHC class I–deficient APCs
APCs, especially macrophages, are the main mediators of TLR­triggered innate inflammatory responses in vivo. Consistent with the
in vivo data presented above, peritoneal macrophages from B2m−/−
mice produced more TNF, IL-6 and IFN-β than did those from
their littermates (control mice) in response to stimulation with LPS,
poly(I:C) or CpG ODN (Fig. 3a and Supplementary Fig. 2a). We
obtained similar results with KbDb−/− macrophages (Supplementary
Fig. 1d). We also found exacerbated cytokine production in
VOLUME 13 NUMBER 6 JUNE 2012 nature immunology
Articles
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CD8+ T cells attenuate TLR responses via MHC class I
To determine the physiological relevance of our observation that
reverse signals from MHC class I molecules suppressed TLR-triggered
immune responses, we looked for cells in vivo that provided ligands
for MHC class I molecules on APCs. The known natural ligands for
MHC class I in vivo are TCRs, the coreceptor CD8, the lectin-like
receptor CD94-NKG2 and killer immunoglobulin-like receptors
expressed mainly on NK cells and CD8+ T cells, which made these
PB
**
4
IFN-β (ng/ml)
B2m–/–
IFN-β (ng/ml)
B2m+/+
5
IL-6 (ng/ml)
a
Ligation of MHC class I molecules via monoclonal antibodies
induces downstream signals in lymphocytes and elicits biological functions13–15. After stimulation with LPS, poly(I:C) or CpG ODN, macrophages crosslinked with antibody to H-2Kb (anti-H-2Kb) secreted less
proinflammatory cytokines and IFN-β than did macro­phages treated
with control antibody (Fig. 3e). Crosslinkage of another MHC class
I molecule, H2-Db, also suppressed the TLR-triggered inflammatory responses in macrophages (Supplementary Fig. 2c), whereas
crosslinkage of both H-2Kb and H2-Db resulted in even less production of these cytokines (data not shown). These data suggested that
crosslinkage of MHC class I molecules exerted an inhibitory effect on
TLR-triggered inflammatory responses in macrophages.
IL-6 (ng/ml)
MHC class I–­deficient bone marrow–derived dendritic cells
(Supplementary Fig. 2b). We then investigated the effect of knockdown of MHC class I on cytokine expression in macrophages in
which TLRs were triggered. Macrophages in which the gene encoding H-2Kb was silenced by H-2Kb-specific small interfering RNA
(siRNA) produced significantly more proinflammatory cytokines
and IFN-β in response to stimulation with LPS, poly(I:C) or CpG
ODN than did those transfected with control siRNA (Fig. 3b,c). To
further demonstrate that the enhanced TLR-triggered inflammatory response in MHC class I–deficient mice in vivo was due to the
MHC class I deficiency in macrophages, we adoptively transferred
MHC class I–deficient macro­phages into wild-type mice depleted
of endogenous macrophages via pretreatment with clodronate
liposomes. After LPS challenge, mice reconstituted with MHC class
I–deficient (B2m−/− or KbDb−/−) macrophages produced more proinflammatory cytokines and IFN-β than did those ­reconstituted with
wild-type macrophages (Fig. 3d and data not shown). Therefore,
these data suggested that MHC class I deficiency enhanced TLRtriggered inflammatory responses in macrophages and that this may
have resulted in the greater susceptibility of MHC class I–deficient
mice to lethal challenge with LPS observed above.
TNF (ng/ml)
npg
© 2012 Nature America, Inc. All rights reserved.
Figure 2 MHC class I–deficient mice are more susceptible to infection with E. coli but are more resistant to infection with L. monocytogenes (LM).
(a,b) ELISA of TNF, IL-6 and IFN-β in serum (a) and analysis of bacterial loads in blood (b) of B2m+/+ and B2m−/− mice (n = 4 per genotype) 2, 4 or
8 h (horizontal axes) after intraperitoneal infection with 1 × 10 8 E. coli. CFU, colony-forming units. (c,d) ELISA of TNF, IL-6 and IFN-β in serum (c) and
analysis of bacterial loads in spleen and liver (d) of B2m+/+ and B2m−/− mice (n = 4 per genotype) after treatment with PBS or infection with 1 × 10 4
L. monocytogenes; serum was obtained 4 h after infection. In b,d, each symbol represents an individual mouse; small horizontal lines indicate the mean.
*P < 0.05 and **P < 0.01 (Student’s t-test). Data are from three independent experiments with similar results (mean ± s.e.m.).
Figure 3 MHC class I reverse signaling inhibits TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages. (a) ELISA
of TNF, IL-6 and IFN-β in supernatants of B2m+/+ and B2m−/− macrophages stimulated for 4 h with PBS, LPS, poly(I:C) or CpG ODN. (b) Immunoblot
analysis of MHC class I (MHCI) in macrophages 48 h after transfection with control (Ctrl) siRNA or siRNA specific for MHC class I. β-actin serves as
a loading control throughout. (c) ELISA of TNF, IL-6 and IFN-β in macrophages transected with siRNA as in b (key), then stimulated for 4 h with LPS,
poly(I:C) or CpG. (d) ELISA of TNF, IL-6 and IFN-β in serum from wild-type mice (n = 4 per group) depleted of endogenous macrophages and then given
adoptive transfer of wild-type macrophages (B2m+/+→B2m+/+) or MHC class I–deficient macrophages (B2m−/−→B2m+/+), followed by LPS challenge
and analysis 2 h later. (e) ELISA of TNF, IL-6 and IFN-β in macrophages after ligation with control antibody (immunoglobulin G (IgG)) or monoclonal
antibody to MHC class I (H-2Kb) and stimulation for 4 h with PBS, LPS, poly(I:C) or CpG ODN. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are
from three independent experiments (a,c–e; mean ± s.e.m.) or are representative of three independent experiments with similar results (b).
nature immunology VOLUME 13 NUMBER 6 JUNE 2012
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LPS
LPS
TNF (ng/ml)
0.5
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–
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0
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MΦ
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3
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**
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NK cell depletion
1.5
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IL-6 (ng/ml)
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CD8+ T cell depletion
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WT
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a
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TNF (ng/ml)
Articles
1
* *
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MΦ
CD8+ T cell
Transwell
Anti–IL-10
Anti–TGF-β
+
–
–
–
–
+
+
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+
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–
+
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Figure 4 CD8+ T cells suppress TLR-triggered production of inflammatory cytokines in macrophages
**
3
8
**
2
in an MHC class I–dependent manner. (a) ELISA of TNF, IL-6 and IFN-β in the serum of wild-type
*
* NS
mice (n = 4 per group) left undepleted (WT) or depleted of CD8 + T cells or NK cells, followed by
6
2
LPS challenge and analysis 2 h later. (b) ELISA of TNF, IL-6 and IFN-β in supernatants of B2m+/+
or B2m−/− macrophages cultured alone (MΦ) or at a ratio of 1:1 with CD8+ T cells (MΦ + CD8+
4
1
T cells) and simulated for 4 h with LPS. (c) ELISA of TNF in supernatants of macrophages cultured
1
2
alone (MΦ) or with NK cells (MΦ + NK) and simulated for 4 h with LPS. (d) ELISA of TNF in
wild-type macrophages cultured in the presence (+) or absence (−) of CD8 + T cells, anti–IL-10
0
0
0
and anti–TGF-β, with (+) or without (−) a Transwell system (to separate CD8 + T cells in the upper
+/+
−/−
chamber), then stimulated for 4 h with LPS (100 ng/ml). (e) TNF in serum from B2m or B2m
mice (left) or B2m−/− mice given transfer of wild-type CD8+ T cells (B2m−/− + CD8+; n = 4 mice per
group), assessed 2 h after LPS challenge. (f) ELISA of TNF in macrophages cultured alone (MΦ)
or with CD8+ T cells (MΦ + CD8+) or OT-I cells (MΦ + OT-I) in the presence (+ OVA) or absence of
ovalbumin peptide (amino acids 257–264). (g) ELISA of TNF in wild-type macrophages cultured alone (MΦ) or with naive (MΦ + naive) or memory
(MΦ + mem) CD8+ T cells (sorted by flow cytometry as CD8+CD44lo or CD8+CD44hi cells, respectively) and stimulated for 2 h with LPS (100 ng/ml).
NS, not significant; *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from three independent experiments with similar results (mean ± s.e.m.).
g
npg
© 2012 Nature America, Inc. All rights reserved.
Φ M
M +n Φ
Φ a
+ ive
m
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–
+
cells the most likely candidates. In vivo depletion of NK cells or CD8+
T cells resulted in significantly enhanced production of TNF, IL-6
and IFN-β in mice after LPS challenge (Fig. 4a), consistent with a
published report22. We further cultured macrophages together with
NK cells or CD8+ T cells in vitro and found that culture with CD8+
T cells resulted in significantly impaired production of TNF, IL-6,
IFN-β by LPS-stimulated macrophages, whereas culture with NK cells
did not (Fig. 4b,c).
To demonstrate the underlying mechanisms by which CD8 +
T cells inhibited macrophage inflammatory responses to TLR ligands,
we used a Transwell system and found that the inhibitory effect of
CD8+ T cells was attenuated when the cells were physically separated
(Fig. 4d). In addition, blockade of the inhibitory cytokines IL-10
or TGF-β did not relieve the suppressive effect of CD8+ T cells
(Fig. 4d). Therefore, CD8+ T cell–mediated suppression of the
innate inflammatory responses of macrophages was dependent on
cell-cell contact. To elucidate whether MHC class I molecules were
involved in the process, we cultured CD8+ T cells together with
MHC class I–deficient macrophages. Notably, the suppressive effect
of CD8+ T cells was completely abrogated (Fig. 4b). Additionally,
when we adoptively transferred wild-type CD8 + T cells into
B2m−/− mice whose macrophages lack expression of MHC class I,
we observed no significant attenuation of inflammatory responses
after LPS stimulation (Fig. 4e). These data suggested a nonredundant
role for MHC class I expression on macrophages in the suppression
of innate responses by CD8+ T cells.
We further determined whether engagement of TCRs by peptide–
MHC class I complexes was required. OT-I cells (which transgenically
express an ovalbumin-specific TCR) tempered cytokine production
by macrophages just as nontransgenic CD8+ T cells did in the absence
of cognate peptide (Fig. 4f). However, after the addition of ovalbumin
peptide, the suppressive effect of OT-I cells was abrogated, whereas
the inhibitory effect of nontransgenic CD8+ T cells was not influenced
by the addition of ovalbumin peptide (Fig. 4f and data not shown). We
also found that naive CD8+ T cells had a greater effect than memory
T cells had in suppressing cytokine production by macrophages in
which TLRs were triggered (Fig. 4g). Together these data suggested
554
that naive CD8+ T cells suppress innate inflammatory responses in
macrophages in an MHC class I–dependent way.
Enhanced TLR signaling in MHC class I–deficient macrophages
To investigate whether MHC class I deficiency intersected with the
TLR signaling pathways in macrophages, we examined the activation kinetics of the mitogen-activated protein kinase (MAPK) and
transcription factor NF-κB pathways, which are both downstream of
TLR signaling. Activation of the MAPKs Jnk, Erk and p38, the kinases
IKKα and IKKβ and the NF-κB inhibitor IκBα was enhanced in LPSstimulated MHC class I–deficient macrophages (Fig. 5a and data not
shown). We also observed more phosphorylation of the transcription
factor IRF3 (Fig. 5a). We obtained similar results with MHC class
I–deficient macrophages stimulated with poly(I:C) or CpG ODN
(Supplementary Fig. 3a). These data suggested that deficiency in
MHC class I enhances TLR signaling in macrophages.
a
B2m+/+
B2m–/–
LPS (min) 0 15 30 45 60 0 15 30 45 60
p-Erk
p-Jnk
p-p38
b
IgG
H-2Kb
LPS (min) 0 15 30 45 60 0 15 30 45 60
p-Erk
p-Jnk
p-p38
p-IKKα-IKKβ
p-IKKα-IKKβ
p-IκBα
p-IκBα
β-actin
β-actin
B2m+/+
B2m–/–
LPS (min) 0 30 60 90 120 0 30 60 90 120
p-IRF3
β-actin
IgG
H-2Kb
LPS (min) 0 30 60 90 120 0 30 60 90 120
p-IRF3
β-actin
Figure 5 MHC class I reverse signaling impairs TLR pathways in
macrophages. (a) Immunoblot analysis of phosphorylated (p-) signaling
molecules in lysates of B2m+/+ and B2m−/− macrophages stimulated
for 0–120 min (above lanes) with LPS. (b) Immunoblot analysis of
phosphorylated signaling molecules in lysates of macrophages treated with
control antibody (IgG) or crosslinked with monoclonal antibody to MHC
class I (H-2Kb) and stimulated with LPS. Data are from one experiment
representative of three independent experiments with similar results.
VOLUME 13 NUMBER 6 JUNE 2012 nature immunology
Articles
a
PM
Cyt
b
c
LPS (min) 15 0 5 15
15 0 5 15
LPS (min) 15 0 5 15
IP: MHCI
– + + +
– + + +
IP: Fps
– + + +
IP: IgG
+ – – –
+ – – –
IP: IgG
+ – – –
IB: Fps
IB: MHCI
IB: MHCI
IB: Fps
MHCI
Fps
Merge
d
Mock
Flag-H2K(WT)
Flag-H2K(del)
Flag-H2K(mut)
Myc-Fps
IP: Flag
LPS
(0 min)
–
+
–
–
+
+
–
–
+
–
+
+
–
–
–
+
+
+
+
–
–
–
+
+
e
Mock
Myc-Fps
Myc-Fps∆SH2
Flag-H2K
IP: Myc
IB: Myc
LPS
(5 min)
–
+
–
+
+
–
–
+
+
+
+
–
–
+
+
IB: Flag
IB: Myc
IB: Flag
KbDb+/+
f
K D
3
Figure 6 Membrane MHC class I molecules interact directly with Fps.
8 *
5 *
* *
*
* *
* *
(a) Immunoassay of lysates of peritoneal macrophages stimulated for
2
4
6
3
0, 5 or 15 min with LPS, followed by immunoprecipitation (IP +) of
4
1
2
plasma-membrane (PM) or cytoplasmic (Cyt) proteins with IgG or
2
1
anti–MHC class I and immunoblot analysis (IB) with anti-Fps or
0
0
0
anti–MHC class I. (b) Immunoassay of lysates of macrophages
stimulated for 0, 5 or 15 min with LPS, followed by immuno­precipitation
with IgG or anti-Fps and immunoblot analysis with anti–MHC class I
or anti-Fps. (c) Confocal microscopy of macrophages stimulated for 0 or 5 min with LPS and labeled with anti–MHC class I and anti-Fps. Original
magnification, ×600. (d) Immunoassay of lysates of macrophages mock transfected (Mock +) or transfected to express Flag-tagged wild-type H-2K
(Flag-H2K(WT)), H-2K lacking the intracellular domain (Flag-H2K(del)) or H-2K with substitution of the intracellular tyrosine site (Flag-H2K(mut)),
plus Myc-tagged Fps (Myc-Fps), then stimulated with LPS, followed by immunoprecipitation with anti-Flag and immunoblot analysis with anti-Myc or
anti-Flag. (e) Immunoassay of lysates of macrophages mock transfected or transfected to express Myc-tagged wild-type Fps (Myc-Fps) or mutant Fps
with deletion of the SH2 domain (Myc-Fps∆SH2), plus Flag-tagged wild-type H-2K, then stimulated with LPS, followed by immunoprecipitation with
anti-Myc and immunoblot analysis as in d. (f) ELISA of TNF, IL-6 and IFN-β in supernatants of KbDb+/+ and KbDb−/− macrophages mock transfected or
transfected to express wild-type H-2K or the H-2K mutants in d. *P < 0.01 (Student’s t-test). Data are representative of three independent experiments
with similar results (a–e) or are from three independent experiments (f; mean ± s.e.m.).
npg
© 2012 Nature America, Inc. All rights reserved.
As ligation of MHC class I molecules attenuated TLR-triggered
inflammatory responses, we further investigated the effect of MHC
class I ligation on TLR pathways. Crosslinkage of MHC class I molecules on macrophages resulted in less activation of the MAPK, NF-κB
and IRF3 pathways in response to LPS stimulation (Fig. 5b), whereas
crosslinkage alone did not activate any of these pathways (data not
shown). We obtained similar results after stimulation with poly(I:C)
or CpG ODN (Supplementary Fig. 3b). Collectively, these data indicated that ligation of MHC class I molecules may have inhibited the
TLR-triggered production of proinflammatory cytokines and type I
interferon by impairing activation of MyD88- and TRIF-dependent
pathways in macrophages.
Membrane MHC class I molecules bind Fps
We further explored the underlying mechanisms of the suppression of TLR signaling by MHC class I molecules. First, we sought
to determine whether MHC class I molecules interacted directly
with TLRs and served as cofactors in inhibiting TLR signaling by
forming a complex. Immunoprecipitation with anti–MHC class
I demonstrated no such interaction between TLRs and MHC
class I molecules, and we obtained similar results by coimmunoprecipitation with anti-TLR as well (data not shown). Confocal
microscopy also showed that the distribution of TLR3, TLR4 and
TLR9 in endosomes in B2m−/− macrophages was similar to that in
wild-type macrophages after stimulation with the ligands for these
TLRs (Supplementary Fig. 4a–c). The downregulation of surface
TLR4 in response to stimulation with LPS was also similar in wildtype and MHC class I–deficient macrophages (Supplementary
Fig. 4d). These results demonstrated that deficiency in MHC class
I did not affect the endosomal distribution of TLR4, TLR3 or TLR9
in macrophages after activation of the TLR, which suggested the
existence of other molecules that interact with MHC class I and
temper TLR signaling.
Given that activation of many tyrosine kinases is involved in the
control of TLR signaling, we immunoprecipitated proteins from
lysates of LPS-stimulated macrophages with anti–MHC class I and
nature immunology VOLUME 13 NUMBER 6 JUNE 2012
IFN-β (ng/ml)
o
2K ck
(W
T
H
2K )
(d
el
H
2K )
(m
ut
)
M
H
M
H ock
2K
(W
T
H
2K )
(
H del
2K )
(m
ut
)
H
M
o
2K ck
(W
T
H
2K )
(
H del)
2K
(m
ut
)
IL-6 (ng/ml)
TNF (ng/ml)
b b–/–
then used reverse-phase nanospray liquid chromatography–tandem
mass spectrometry to identify possible MHC class I–associated tyrosine kinases or other molecules that may be involved in the suppression of TLR pathways. Among the kinases shown to be associated with
MHC class I, the non-receptor kinase Fps attracted our attention, as
Fps-deficient mice have been shown to be more susceptible to LPSinduced sepsis23. An immunoprecipitation assay confirmed that Fps
did indeed interact with MHC class I molecules at steady state, and
this interaction increased after LPS stimulation (Fig. 6a,b). In addition, plasma-membrane MHC class I molecules associated with Fps,
but intracellular MHC class I molecules did not (Fig. 6a). Confocal
microscopy showed a dispersed pattern of Fps in the cytosol of
unstimulated macrophages, although small amounts associated with
MHC class I. After stimulation with TLR ligands, Fps was activated
and aggregated as clusters along the cytoplasmic side of the membrane
and showed substantially enhanced colocalization with membrane
MHC class I molecules (Fig. 6c).
MHC class I binds Fps via intracellular phosphorylated tyrosines
We went further to determine which domains of MHC class I and
Fps were required for their interaction and for the suppression of
TLR-triggered responses. Distinct from MHC class II molecules,
MHC class I molecules have a relatively long tail and a tyrosinephosphorylation site in the cytoplamic domain (Tyr320 in human
and Tyr321 in mice), whereas Fps contains a Src-homology 2 (SH2)
domain. Thus, we hypothesized that these two molecules may interact
directly. We transfected mouse macrophages to express Myc-tagged
Fps together with Flag-tagged wild-type H-2K, H-2K lacking the
intra­cellular domain or H-2K with substitution of the intracellular
tyrosine site. Coimmunoprecipitation showed that Fps interacted
with wild-type MHC class I molecules but not with those with a
mutant intra­cellular domain or tyrosine site (Fig. 6d). Mutant Fps
with deletion of the SH2 domain did not interact with MHC class I
either (Fig. 6e), which suggested a nonredundant role for the SH2
domain in the interaction of Fps and MHC class I. Gain-of-function ­experiments also showed that overexpression of wild-type MHC
555
0
**
4
3
2
1
0
**
**
2.5
2.0
1.5
1.0
0.5
0
**
**
**
S
Po LP
ly S
(I:
C
C )
pG
1
*
PB
**
S
Po LPS
ly
(I:
C
)
C
pG
2
Fps
IL-6 (ng/ml)
**
PB
siRNA:
Fps
β-actin
Ctrl
3
S
Po LP
ly S
(I:
C
C )
pG
IP: Fps
d
PB
IP: Fps
LPS (min)
p-Fps
Fps
c
H-2Kb
0
15
30
45
60
0
15
30
45
60
0
15
30
45
60
0
15
30
45
60
LPS (min)
p-Fps
Fps
lgG
B2m–/–
TNF (ng/ml)
b
B2m+/+
C
trl
Fp
s
a
IFN-β (ng/ml)
Articles
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© 2012 Nature America, Inc. All rights reserved.
class I ­ significantly suppressed the production of TNF, IL-6 and
IFN-β in KbDb−/− macrophages, whereas overexpression of mutant
MHC class I did not have this effect (Fig. 6f). Therefore, the tyrosine
site of MHC class I and the SH2 domain of Fps were required for their
interaction and for the suppressive effect of MHC class I molecules on
TLR-triggered responses.
MHC class I suppresses TLR signaling via Fps activation
As tyrosine-phosphorylation of the intracellular domain of MHC
class I was required for binding to the SH2 domain of Fps, we further
determined which signaling molecules contributed to this phosphor­
ylation. The kinase Src is suggested to phosphorylate MHC class I
molecules in Jurkat human T lymphocyte cells24. It has been shown
that Src is activated in macrophages after stimulation with TLR
­ligands20 and is able to restrain TLR-triggered cytokine production. Suppression of Src activity with its inhibitor PP1 substantially
suppressed the phosphorylation of MHC class I and resulted in less
Fps–MHC class I association in macrophages after stimulation with
LPS (Supplementary Fig. 5a,b). Fps activation was also much lower
when Src activity was inhibited (Supplementary Fig. 5c), which suggested a nonredundent role for Src in the interaction of MHC class I
and Fps and also in the activation of Fps. Thus, Src may be involved
in phosphorylation of the cytoplamic tail of MHC class I, allowing the association of MHC class I with Fps, which then transduces
downstream signals.
To investigate the exact role of Fps in the negative regulation of
TLR signaling by MHC class I molecules, we examined the activation of Fps after stimulation with TLR ligands. Indeed, there was
much less TLR ligand–induced phosphorylation of Fps in B2m−/−
or KbDb−/− macrophages (Fig. 7a and data not shown). Crosslinkage
of MHC class I molecules further enhanced their interaction with
Fps and phosphorylation of Fps (Fig. 7b and Supplementary Fig.
6), which indicated that MHC class I molecules may have increased
or maintained the TLR-triggered activation of Fps. Silencing of Fps
resulted in more production of TNF, IL-6 and IFN-β in macrophages
stimulated with LPS, poly(I:C) or CpG ODN (Fig. 7c,d), which suggested a negative role for Fps in TLR responses. We also found that
knockdown of Fps was less potent in suppressing the TLR-triggered
production of cytokines in B2m−/− macrophages than it was in
wild-type macrophages (Fig. 7e). Accordingly, the ­inhibitory effect
of crosslinkage of MHC class I molecules on TLR signaling was also
substantially attenuated in macrophages in which the gene encoding
Fps was silenced (data not shown). Together these data suggested
556
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
C
trl
Fp
s
IL-6 (ng/ml)
TNF (ng/ml)
e
IFN-β (ng/ml)
Figure 7 MHC class I molecules suppress TLR-triggered response by
B2m–/–
B2m+/+
maintaining Fps activation. (a,b) Immunoassay of B2m+/+ and B2m−/−
*
*
macrophages (a) or wild-type macrophages crosslinked with IgG or
2.5
3
6
* ***
*
*
**
5 **
2.0 **
*
anti–H-2Kb (b), stimulated for 0–60 min (above lanes) with LPS, followed
*
**
**
2
4
1.5
**
**
by immunoprecipitation with anti-Fps and immunoblot analysis of
3
**
1.0
1
2
phosphorylated Fps (p-Fps; detected by probing for phosphorylated tyrosine)
0.5
1
0
0
0
or total Fps. (c) Immunoblot analysis of Fps in lysates of macrophages
siRNA:
siRNA:
siRNA:
transfected for 48 h with control or Fps-specific siRNA. (d,e) ELISA
LPS Poly CpG
LPS Poly CpG
LPS Poly CpG
of TNF, IL-6 and IFN-β in supernatants of wild-type C57BL/6 macrophages (d)
(I:C)
(I:C)
(I:C)
or B2m+/+ and B2m−/− macrophages (e) transfected with control or Fps-specific
siRNA and stimulated for 4 h with PBS, LPS, poly(I:C) or CpG ODN. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three
independent experiments with similar results (a–c) or are from three independent experiments (d,e; mean ± s.e.m.).
that membrane MHC class I molecules suppressed TLR-triggered
inflammatory responses by associating with Fps and maintaining
its activation.
Fps suppresses TLR-triggered response by activating SHP-2
Fps activates the cell-adhesion molecule PECAM-1 (CD31) to mediate the suppressive effect of the receptor FcεRI on mast-cell activation25, and published data have suggested that PECAM-1 can recruit
SHP-2 to suppress TLR4 signaling26. Accordingly, we found that activation of SHP-2 in B2m−/− or KbDb−/− macrophages was considerably
attenuated after stimulation with LPS (Fig. 8a and data not shown),
which indicated that SHP-2 may have been involved in the suppression of TLR signaling by MHC class I molecules. Deficiency in SHP-2
enhanced the production of proinflammatory cytokines and IFN-β in
macrophages stimulated with LPS, poly(I:C) or CpG ODN (Fig. 8b).
However, the inhibitory effect of crosslinkage of MHC class I on
TLR-triggered cytokine production was considerably attenuated in
SHP-2-deficient macrophages (Fig. 8c). These data indicated that
activation of SHP-2 might have contributed to the suppression of
TLR signaling in macrophages by MHC class I molecules.
Next we investigated whether Fps contributed to the activation of
SHP-2 enhanced by MHC class I molecules. After stimulation with
TLR ligands, the phosphorylation of SHP-2 was considerably attenuated in macrophages in which the gene encoding Fps was silenced
(Fig. 8d), which suggested that SHP-2 functions downstream of
Fps. To more convincingly demonstrate the link between SHP-2 and
Fps, we did coimmunoprecipitation assays and found an association
between SHP-2 and Fps in resting macrophages, which was enhanced
by stimulation with LPS (Fig. 8e). However, MHC class I molecules
did not directly interact with SHP-2 (data not shown).
SHP-2 suppresses TLR-triggered TRIF-dependent signal pathways by binding to and inhibiting activation of the kinase TBK1
(ref. 27). However, the molecules that interact with SHP-2 in the
MyD88-dependent pathway have not been identified so far. As
activation of both NF-κB and MAPKs was enhanced in MHC class
I–­deficient macrophages, we investigated whether SHP-2 interacted
with MyD88, the signaling molecule TRAF6, the kinase IRAK1
and other molecules involved in early events in TLR signaling.
Among those, only TRAF6 interacted with SHP-2, a result confirmed by reverse coimmuno­precipitation (Fig. 8f). The carboxyl
terminus of SHP-2 was responsible for its interaction with TRAF6
(Supplementary Fig. 7a). A luciferase reporter assay also showed
that SHP-2 ­suppressed TRAF6-triggered activation of NF-κB in a
VOLUME 13 NUMBER 6 JUNE 2012 nature immunology
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b
© 2012 Nature America, Inc. All rights reserved.
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f
dose-dependent manner (Supplementary Fig. 7b). As ubiquitination of TRAF6 is required in MyD88-dependent signaling, we
further compared the ubiquitination of TRAF6 in wild-type and
SHP-2-­deficient macrophages. SHP-2 deficiency resulted in more
LPS-induced polyubiquitination of TRAF6 (Fig. 8g), which indicated that SHP-2 suppressed the MyD88 pathway by interacting with
TRAF6 and controlling its ubiquitination. Collectively, these data
suggested that MHC class I molecules negatively regulated TLRtriggered inflammatory responses by enhancing activation of the
Fps–SHP-2 pathway (Supplementary Fig. 8).
DISCUSSION
Reverse signaling by members of the TNF family28 (including FasL,
4-1BBL, CD40L, LIGHT, membrane TNF and so on) and the B7
­family29,30 (including CD80, CD86, B7-H1, B7-DC and so on) has
been under extensive exploration. Reverse signaling mediated by MHC
class I molecules on the cell surface has also been demonstrated11–15,
but there have been no reports on this signaling and its subsequent
effects on myeloid cells. Here we have provided evidence that MHC
class I molecules on macrophages stimulated with TLR ligands and/or
after ligation with certain monoclonal antibodies may interact directly
with Fps, maintaining its phosphorylation. Fps then recruits SHP-2,
and SHP-2 participates at least partially in the suppression of TLR signaling by MHC class I molecules. Therefore, we have demonstrated a
previously unknown nonclassical function for MHC class I molecules
in which they are involved, via reverse signaling, in the negative regulation of TLR-triggered innate inflammatory responses.
Adaptive T cells are able to temper innate immune responses;
however, details of the mechanisms involved are unknown31. In the
nature immunology VOLUME 13 NUMBER 6 JUNE 2012
S
ly
(I:
C
)
C
pG
Po
LP
IFN-β (ng/ml)
PB
S
IFN-β (ng/ml)
S
L
Po PS
ly
(I:
C
)
C
pG
PB
S
ly
(I:
C
)
C
pG
IL-6 (ng/ml)
TNF (ng/ml)
c
d
LP
Po
PB
S
IL-6 (ng/ml)
TNF (ng/ml)
Figure 8 Binding of SHP-2 to Fps and
Shp2fl/fl
Shp2–/–
2.5
5
3
**
enhanced activation of SHP-2 are required
+/+
–/–
**
**
B2m
B2m
2.0
**
4
**
for negative regulation of TLR responses by
**
**
2
**
LPS (min) 0 15 30 45 60 0 15 30 45 60
1.5
3
**
MHC class I molecules. (a) Immunoblot
1.0
2
p-SHP-2
1
analysis of total and phosphorylated SHP-2
0.5
1
in lysates of B2m+/+ and B2m−/− peritoneal
SHP-2
0
0
0
macrophages stimulated for 0–60 min (above
lanes) with LPS. (b) ELISA of TNF, IL-6
and IFN-β in supernatants of macrophages
6
3
4
IgG
H-2Kb
with loxP-flanked alleles encoding SHP-2
5
(Ptpn11fl/fl; called ‘Shp2fl/fl’ here) and
3
4 **
2
**
**
**
SHP-2-deficient macrophages (Ptpn11−/−;
*
**
**
**
3
2
called ‘Shp2−/−’ here) stimulated for 4 h
**
1
2
with PBS, LPS, poly(I:C) or CpG ODN.
1
1
(c) ELISA of TNF, IL-6, IFN-β in supernatants
0
0
0
of macrophages of the genotypes in b, treated
Shp2 fl/fl –/– fl/fl –/– fl/fl –/–
Shp2 fl/fl –/– fl/fl –/– fl/fl –/–
Shp2 fl/fl –/– fl/fl –/– fl/fl –/–
b
with IgG or anti–H-2K (for crosslinking
LPS
Poly(I:C)
CpG
LPS
Poly(I:C)
CpG
LPS Poly(I:C) CpG
of MHC class I) and stimulated as in b.
LPS (min) 15 0 5 15
LPS (min) 15 0 5 15
(d) Immunoblot analysis of phosphorylated
Ctrl siRNA
Fps siRNA
IP: SHP-2 – + + +
IP: Fps – + + +
SHP-2 in wild-type macrophages transfected
LPS (min) 0 15 30 45 60 0 15 30 45 60
IP: IgG + – – –
IP: IgG + – – –
with control or Fps-specific siRNA and then
p-SHP-2
IB: SHP-2
IB: Fps
stimulated for 0–60 min (above lanes) with
IB: Fps
IB: SHP-2
SHP-2
LPS. Total SHP-2 serves as loading control.
(e) Immunoblot analysis of Fps and SHPShp2fl/fl
Shp2–/–
2 in lysates of macrophages stimulated for
LPS (min)
0 15 30 60 0 15 30 60 (kDa)
LPS (min) 15 0 15 30 60
LPS (min) 15 0 15 30 60
170
0, 5 or 15 min with LPS and subjected to
IP: TRAF6 – + + + +
IP: SHP-2 – + + + +
130
immunoprecipitation with IgG or anti–SHP-2
IP:
TRAF6
IP: IgG + – – – –
IP: IgG + – – – –
95
(left) or anti-Fps (right). (f) Immunoblot
IB: Ub
IB: SHP-2
IB: TRAF6
72
analysis of TRAF6 and SHP-2 in lysates of
IB:
SHP-2
IB: TRAF6
LPS-stimulated macrophages subjected
IP: TRAF6
to immunoprecipitation with IgG or
IB: TRAF6
anti-TRAF6 (left) or anti–SHP-2 (right).
(g) Immunoblot analysis of lysates of LPS-stimulated macrophages (genotypes, as in b) subjected to immunoprecipitation with anti-TRAF6 and probed
with anti-ubiquitin (Ub; above) or anti-TRAF6 (below). kDa, kilodaltons. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three
independent experiments with similar results (a,d–g) or are from three independent experiments (b,c; mean ± s.e.m.).
e
g
clinical context, patients with type I bare lymphocyte syndrome
(also known as TAP-deficiency syndrome) lack almost all surface
expression of MHC class I molecules and CD8+ T cells32,33. These
patients have high concentrations of C-reactive protein and a high
erythrocyte-­sedimentation rate during acute infection, which suggests they have enhanced innate inflammatory responses33. We indeed
found that CD8+ T cells suppressed the macrophage response to TLRs
both in vitro and in vivo via MHC class I molecules on macrophages.
However, we did not obtain details about the exact ligands of MHC
class I on CD8+ T cells, which may be CD8, TCRαβ or one of the
NK cell–related inhibitory receptors. Furthermore, other ligands or
receptors for MHC class I molecules expressed on monocytes and
macrophages have been suggested, such as the inhibitory receptors
ILT-2 and ILT-4 (ref. 34). During activation in vitro or in vivo, those
MHC class I ligands, and even unknown ligands, may interact in cis or
in trans with MHC class I molecules on the membrane, providing the
reverse signal to MHC class I molecules, and, finally, may orchestrate
the inhibitory effect of MHC class I on TLR signaling.
Accumulating evidence suggests that a pool of MHC class I molecules present at the plasma membrane can dissociate from the
light-chain β2m and open their structure, resulting in MHC class I
molecules known as ‘open conformers’ or ‘misfolding conformers’35.
These are sparse in quiescent cells but increase considerably in abundance when the cells are metabolically activated. The open conformers
are not stable and tend to associate with certain ­receptors or even with
themselves, which is believed to be the basis for the reverse ­signaling
of the biological effect of MHC class I molecules35. Notably, the cytoplasmic tail of MHC class I, which is somewhat longer than that of
MHC class II, has one conserved tyrosine residue, which provides a
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© 2012 Nature America, Inc. All rights reserved.
Articles
recognition site for the SH2 domain of kinases11,24. In addition, phosphorylation of the tyrosine residue in the cytoplasmic domain of MHC
class I molecules is also related to the formation of open conformers and may influence or reflect interaction with certain molecules
and the initiation of MHC class I reverse-signaling pathways24,35,36.
Moreover, Fps can be activated after stimulation by certain cytokines,
such as GM-CSF, IL-4, IL-6, erythropoietin and so on, and has been
detected as being associated with phosphorylated receptors of these
cytokines, including IL-4Rα, gp130 and the ­common β-chain37–39.
Therefore, we predicted and have proven that phosphorylation of
Tyr321 of intracellular MHC class I was necessary for its association
with the SH2 domain of Fps and that this interaction may contribute
to maintenance of Fps activation and the suppressive function of Fps
on TLR-triggered responses40.
Fps is a member of the non-receptor tyrosine kinase family, which
has critical roles in regulating cytoskeletal rearrangements and the
inside-out signaling involved in receptor-ligand, cell-matrix and cellcell interactions41,42. Fps is expressed in hematopoietic cells of the
myeloid lineage, including macrophages, neutrophils and so on, and
is essential for the survival and terminal differentiation of myeloidprogenitor cells. Mice deficient in Fps expression are hyper-responsive
to endotoxin challenge23, although the underlying mechanisms have
not been fully elucidated. Here we have shown that Fps associated
with SHP-2 and that this association had a nonredundant role in the
suppression of TLR responses by MHC class I molecules.
SHP-2 is a non-receptor tyrosine phosphatase whose ubiquitous
expression pattern is similar to that of MHC class I molecules 43.
SHP-2 functions as a negative regulator in various signaling pathways through its phosphatase activity. Here, SHP-2 inhibited TLR
signaling by binding to and controlling the activation of TRAF6
and TBK1 of the MyD88-dependent pathway and TRIF-dependent
pathway27, respectively. In addition to SHP-2, Fps may also suppress
TLR signaling via other mechanisms. It has been reported that Fpsdeficient macrophages have impaired internalization of TLR4 when
stimulated with LPS44, which may provide another explanation for
the Fps-­mediated suppression.
In conclusion, our results have demonstrated that membrane MHC
class I molecules interacted with Fps and subsequently induced SHP-2
activation in response to stimulation with TLR ligands and, finally,
suppressed innate inflammatory responses. Therefore, constitutively
expressed MHC class I molecules are required for maintenance of the
quiescent state and fine tuning of TLR-triggered innate inflammatory
responses in macrophages. Our findings provide new insight into
the negative regulation of TLR signaling and indicate a previously
unidentified nonclassical function for MHC class I molecules in the
regulation of innate inflammatory responses.
Methods
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary information is available in the online version of the paper.
Acknowledgments
We thank G. Feng (University of California, San Diego) for mice with
loxP-flanked alleles encoding SHP-2; H. Shen (University of Pennsylvania
School of Medicine) for L. monocytogenes; N. van Rooijen (Free University of
Amsterdam) for liposomes; J. Long, X. Zuo and P. Ma for technical assistance;
and T. Chen and Y. Han for discussions. Supported by the National Key Basic
Research Program of China (2012CB910202 and 2010CB911903), the National
125 Key Project (2012ZX10002-014 and 2012AA020901), the National Natural
Science Foundation of China (81123006) and the Shanghai Committee of Science
and Technology (10DZ1910300).
558
AUTHOR CONTRIBUTIONS
X.C. and S.X. designed the experiments; S.X., X.L., Y.B., C.H., X.Zhu, P.Z.,
W.L. and X.Zha. did the experiments; X.C., S.X. and X.L. analyzed data and
wrote the paper; and X.C. was responsible for research supervision, coordination
and strategy.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/ni.2283.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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ONLINE METHODS
Mice and reagents. C57BL/6J mice were from Joint Ventures Sipper BK
Experimental Animals. Mice deficient in β2m (B6.129P2-B2mtm1Unc/J;
002087), OT-I mice (Tg(TcraTcrb)1100Mjb/J; 003831) and Mx-Cre mice
(B6.Cg-Tg(Mx1-cre)1Cgn/J; 003556) were from Jackson Laboratories. Mice
deficient in H-2Kb and H-2Db were from Taconic Farms. Shp2fl mice (provided
by G. Feng) were crossed with Mx-Cre mice. Shp2fl/fl mice and Mx-Cre ×
Shp2fl/fl mice were injected intraperitoneally with 250 µg poly(I:C) every
other day for a total of five doses before use. All mice were bred in specific
pathogen–free conditions. All animal experiments were in accordance with
the National Institute of Health Guide for the Care and Use of Laboratory
Animals, with the approval of the Scientific Investigation Board of the Second
Military Medical University, Shanghai. LPS (from E. coli serotype 0111:B4),
CpG ODN and poly(I:C) have been described19. E. coli serotype 0111:B4 was
from the China Center for Type Culture Collection. L. monocytogenes was
provided by H. Shen. Antibody to MHC class I (anti-H-2Kb; AF6-88.5) was
from Biolegend. Anti-TLR4 (ab22048), anti-TLR3 (ab62566) and anti-TLR9
(ab52967) were from Abcam. Antibody to Erk phosphorylated at Thr202Tyr204 (E10), to Jnk phosphorylated at Thr183-Tyr185 (G9), to p38 phosphor­
ylated at Thr180-Tyr182 (9211), to IRF3 phosphorylated at Ser396 (4D4G),
to IκBα phosphorylated at Ser32-36 (5A5), to IKKα-IKKβ phosphorylated at
Ser176-Ser180 (16A6), to SHP-2 phosphorylated at Tyr580 (3703), to SHP-2
(D50F2) and to the Myc tag (2272) were from Cell Signaling Technology.
Anti-Fps (sc-25415), anti-TRAF6 (sc-7221, sc-8409), anti-ubiquitin (P4D1),
anti-IRAK1 (sc-5288) and anti-β-actin (sc-130656) were from Santa Cruz.
Anti-Flag (M2) was from Sigma.
Cell culture and RNA-mediated interference. Thioglycollate-elicited
mouse peritoneal macrophages were prepared and cultured in endotoxin-free
RPMI-1640 medium with 10% FCS (Invitrogen) as described19. The siRNA
targeting MHC class I α-chain or Fps was from Dharmacon. Mouse peritoneal macrophages were transfected with siRNA duplexes through the use of
INTERFERin reagent (Polyplus) according to a standard protocol.
Cytokine detection. TNF, IL-6 and IFN-β in supernatants and serum were
measured with ELISA kits (R&D Systems).
RNA quantification. A LightCycler (Roche) and SYBR RT-PCR kit (Takara)
were used for quantitative real-time RT-PCR analysis as described19. Data were
normalized to β-actin expression.
Immunoprecipitation and immunoblot analysis. Cells were lysed with celllysis buffer (Cell Signaling Technology) supplemented with protease inhibitor
‘cocktail’ (Calbiochem). Protein concentrations of the extracts were measured
with a BCA assay (Pierce). The immunoprecipitation assays and immunoblot
assays were done as described19,20.
Nanospray liquid chromatography–tandem mass spectrometry.
Macrophages (3 × 108) were stimulated for 15 min with LPS with or without
crosslinkage of MHC class I, then were lysed for preparation of immunoprecipitates with anti–MHC class I. Proteins were eluted and digested. Digests were
analyzed by nano-ultra-performance liquid chromatography–electrospray
ionization tandem mass spectrometry19. Data from liquid chromatography–
tandem mass spectrometry were processed through the use of ProteinLynx
nature immunology
Global Serverversion 2.4 (PLGS 2.4); the resulting peak lists were used for
searching the NCBI protein database with the Mascot search engine.
Cell isolation and in vivo depletion. CD8+ T cells and NK cells were sorted
from C57BL/6 or OT-I mice with a MoFlo XDP (DakoCytomation). Naive
CD8+ T cells were sorted as CD44loCD62Lhi and memory CD8+ T cells were
sorted as CD44hiCD62Llo. Cell purity was >97%. For depletion of auto­logous
CD8+ T cells or NK cells in vivo, mice were given monoclonal anti-CD8
(2.43; American Type Culture Collection) or anti-NK1.1 (PK136; American
Type Culture Collection), respectively (each at a dose of 200 µg per mouse),
3 d before infection. Depletion frequency was confirmed as being >90%.
Confocal microscopy. Macrophages, plated on glass coverslips in six-well
plates, were left unstimulated or stimulated with LPS and then labeled with
anti–MHC class I, anti-Fps, anti-TLR4, anti-TLR3, anti-TLR9 or anti-EEA1
(C45B10; Cell Signaling Technology). Cells were observed with a Leica TCS
SP2 confocal laser microscope.
Establishment of the endotoxin-shock model and bacterial infection. The
endotoxin-shock mouse model was established by intraperitoneal injection of
LPS (15 mg per kg body weight (mg/kg)) as described20. Serum TNF, IL-6 and
IFN-β were measured by ELISA in mice given intraperitoneal injection of LPS
(5 mg/kg), poly(I:C) (20 mg/kg) or CpG ODN (20 mg/kg). For bacterial infection, E. coli serotype 0111:B4 and L. monocytogenes in mid-logarithmic growth
were collected, counted on agar plates and then resuspended in PBS. Mice were
given intraperitoneal injection of 1 × 107 E. coli or intravenous injection of
1 × 104 L. monocytogenes. Serum was collected for measurement of cytokines
(or colony-forming units for E. coli) and spleens or livers were lysed for mea­
surement of colony-forming units (L. monocytogenes) as described20,45.
Bone marrow transplantation. Bone marrow cells (1 × 107) from B2m−/−
mice, KbDb−/− mice or their littermates (control mice) were transplanted
via tail-vein injection into lethally irradiated wild-type mice (10 Gy). After
8 weeks, CD8+ T cells in the spleen and lymph nodes were counted and MHC
class I expression on macrophages was analyzed by flow cytometry.
Macrophage reconstitution. Bone marrow cells from B2m−/− mice, KbDb−/−
mice or their littermates (control mice) were cultured for 7 d in mouse macro­
phage colony-stimulating factor (50 ng/ml; PeproTech) for the preparation
of bone marrow–derived macrophages. Clodronate liposomes were injected
intraperitoneally into wild-type recipient mice (50 mg in 200 µl per mouse) for
depletion of endogenous macrophages. Then 2 d later, B2m+/+ or B2m−/− bone
marrow–derived macrophages (1 × 107) were transplanted into the recipient
mice by tail-vein injection 6 h before challenge with LPS.
Statistical analysis. The statistical significance of comparisons between two
groups was determined with Student’s t-test. The statistical significance of
survival curves was estimated according to the method of Kaplan and Meier,
and the curves were compared with the generalized Wilcoxon’s test. P values
of less than 0.05 were considered statistically significant.
45.Xu, S. et al. IL-17A-producing γδT cells promote CTL responses against Listeria
monocytogenes infection by enhancing dendritic cell cross-presentation. J. Immunol.
185, 5879–5887 (2010).
doi:10.1038/ni.2283