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Regular Article
PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
Inability to resolve specific infection generates innate immunodeficiency
syndrome in Xiap2/2 mice
Wan-Chen Hsieh,1,2 Ya-Ting Chuang,3 I-Hsuan Chiang,2,4 Shu-Ching Hsu,5 Shi-Chuen Miaw,4 and Ming-Zong Lai1,2,4
1
Graduate Institute of Life Science, National Defense Medical College, Taipei, Taiwan; 2Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan;
Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan; 4Graduate Institute of Immunology, National Taiwan University,
Taipei, Taiwan; and 5National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan
3
Emerging evidence indicates that innate immunodeficiency syndromes are linked to
mutations in innate receptors and to specific infections. X-linked lymphoproliferative
syndrome type-2 (XLP-2) is associated with deficiency in X-linked inhibitor of apoptosis
• XIAP deficiency selectively
diminishes BCL10-mediated protein (XIAP), with poorly understood molecular mechanisms. Here we showed that
XIAP deficiency selectively impaired B-cell chronic lymphocytic leukemia/lymphoma
innate responses and
did not affect responses
impairs the ability of the host 10 (BCL10)-mediated innate responses to dectin-1 ligands but2/2
to various Toll-like receptor agonists. Consequently, Xiap
mice became highly
to control specific microbes.
vulnerable on Candida albicans infection. The compromised early innate responses led
• The selective innate
to the persistent presence of C albicans and inflammatory cytokines in Xiap2/2 mice.
immunodeficiency in the
Furthermore, priming of Xiap2/2 mice with the dectin-1 ligand curdlan alone resulted in
XIAP-deficient host leads to
XLP-2–like syndromes. Restoration of dectin-1–induced Rac1 activation and phagocythe persistent presence of
tosis by resolvin D1, but not up-regulation of nuclear factor-kB, rescued Xiap2/2 mice
specific pathogens and
from C albicans lethal infection. Therefore, development of XLP-2 in XIAP-deficient
excess inflammation.
patients could be partly due to sustained inflammation as a consequence of defective
BCL10-dependent innate immunity toward specific pathogens. Importantly, our results
suggest the potential therapeutic value of resolvin D1 in the treatment of XLP-2 and innate immunodeficiency syndromes.
(Blood. 2014;124(18):2847-2857)
Key Points
Introduction
Mutations in innate receptors and their signaling molecules lead
to a variety of innate immunodeficiency syndromes.1-4 Patients
with primary innate immunodeficiency are characterized by normal
leukocyte development and susceptibility to specific infection. For
example, interleukin-1 receptor-associated kinase-4 deficiency
reveals its indispensable role to control pyrogenic bacteria but not
most other pathogens, whereas Toll-like receptor (TLR)-3 deficiency
selectively impairs immunity to herpes simplex virus.1,2 Recent
findings suggest that Crohn disease also results from a defective
innate inflammatory response.5-7
X-linked lymphoproliferative syndrome type-2 (XLP-2) is a
lymphoproliferative disease associated with deficiency in the X-linked
inhibitor of apoptosis protein (XIAP).8 XLP-2 manifests as sensitivity
to Epstein-Barr virus infection, leading to hemophagocytic lymphohistiocytosis, an inflammatory disorder caused by excess cytokine
production from overactivated lymphocytes and macrophages.9,10
XLP-2 is also associated with recurrent splenomegaly, fever, and
hemorrhagic colitis.10 The molecular mechanism describing how
XIAP deficiency leads to XLP-2 remains unknown.9
XIAP is the best-characterized member of the inhibitor of
apoptosis protein (IAP) family, and is the only IAP protein that
directly inhibits caspase-3, -7, and -9.11,12 However, Xiap2/2 mice
appear normal and do not exhibit any phenotype reminiscent of
XLP-2.13-15 Notably, Xiap2/2 mice are sensitive to infection of Listeria
monocytogenes, with impaired innate immune responses and defective
activation in macrophages.16 XIAP is also known to activate nuclear
factor (NF)-kB independent of the inactivation of caspase.12,17-19 The
requirement of XIAP in NF-kB activation is stimuli selective, and
XIAP is involved in nonobese diabetic (NOD)2-initiated NF-kB
activation but is also dispensable in tumor necrosis factor (TNF)-a- and
lipopolysaccharide (LPS)-induced NF-kB activation.20-22
Dectin-1 is the receptor recognizing b-glucan and is required for
control of fungal infection.23-27 Ligation of dectin-1 activates protein
tyrosine kinase Syk, followed by activation of protein kinase C-d.27
Protein kinase C-d in turn phosphorylates caspase recruitment
domain 9 (CARD9), leading to formation of the CARD9–B-cell
chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mucosaassociated lymphoid tissue lymphoma translocation 1 (MALT1)
complex and activation of IkB kinase and NF-kB.23,24 Dectin-1
signaling also activates extracellular signal-regulated kinase, c-Jun
N-terminal kinase, and p38 mitogen-activated protein kinase
(MAPK).28
Submitted March 24, 2014; accepted August 18, 2014. Prepublished online as
Blood First Edition paper, September 4, 2014; DOI 10.1182/blood-2014-03564609.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
© 2014 by The American Society of Hematology
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HSIEH et al
In the present study, we demonstrate that XIAP is specifically
required for dectin-1–induced innate responses. XIAP targeted BCL10,
and XIAP deficiency impaired the dectin-1–induced BCL10-mediated
activation signals. Consequently, Xiap2/2 mice were unable to
control infection by Candida albicans. The persistent presence of
C albicans resulted in overactivation of macrophages, excess production of inflammatory cytokines, and splenomegaly in Xiap2/2
mice. Importantly, restoration of Rac1 activation and phagocytosis,
but not NF-kB activation, by resolvin D1 rescued Xiap2/2 mice from
C albicans lethal infection. Our results provide evidence of an
innate immunity-associated mechanism that may contribute to the
generation of XLP-2 syndromes in XIAP mutant patients. Furthermore, we suggest the potential therapeutic value of resolvin D1 in
treating XLP-2 and, possibly, innate immunodeficiency syndromes.
Methods
Reagents and mice
Zymosan depleted (InvivoGen) is the hot alkali-treated cell wall from
Saccharomyces cerevisiae lacking all its endogenous TLR-stimulating activity
but retaining dectin-1–activating capacity. The human XIAP constructs
were previously described.29 The Xiap2/2 mouse14 was a generous gift from
Dr David L. Vaux (Walter and Eliza Hall Institute of Medical Research,
Parkville, Australia). Mice were maintained in the specific pathogen-free
mouse facility of the Institute of Molecular Biology, Academia Sinica. All
mouse experiments were conducted with approval from the Institutional
Animal Care and Use Committee, Academia Sinica. Detailed reagents are
listed in supplemental Methods available on the Blood Web site.
Bone marrow-derived macrophages and human macrophages
Bone marrow cells were collected from tibias and femurs flushed with cold
phosphate-buffered saline through a 24-gauge needle and were cultured
in complete Dulbecco’s modified Eagle medium containing 20 ng/mL
granulocyte macrophage–colony-stimulating factor (R&D) for 8 days to
generate bone marrow-derived dendritic cells or in Dulbecco’s modified Eagle
medium containing 20% L929 cell-conditioned medium to generate bone
marrow-derived macrophages. Human CD141 monocytes were isolated from
peripheral blood mononuclear cells and were differentiated into macrophages
in the presence of macrophage–colony-stimulating factor. Rac1V12 or small
interfering XIAP was introduced into human macrophages by electroporation in a Nucleofector (Amaxa, Koeln, Germany). Human peripheral blood
mononuclear cells were used with approval from the Institutional Review
Board of Biomedical Science Research, National Health Research Institutes.
Yeast preparation and quantitation
C albicans (ATCC90028) was cultured on a yeast mold plate at 25°C for 2
days, and a single colony was inoculated into 10 mL YM broth at 30°C for 18
to 24 hours. C albicans were harvested by centrifugation. The yeast pellet was
resuspended with phosphate-buffered saline and the OD was determined.
For determination of fungal burden in vivo, organs were removed from the
infected mice, ground, and resuspended in water. The samples were serially
diluted, and the diluents were spread onto YM plates. The number of
colonies on plates was counted after incubation at 30°C for 2 days.
Resolvin D1 administration
Aspirin-triggered Resolvin D1 (AT-RvD1; Cayman) (abbreviated as RvD1)
was dissolved in phosphate-buffered saline and was administered intravenously
(100 ng/mouse)30-32 3 days after C albicans infection. For in vitro experiments, bone marrow-derived macrophages (BMDMs) were treated with RvD1
for 30 minutes before the addition of heat-killed C albicans (HKCA).
Additional methods are reported in supplemental Methods.
Results
XIAP mediates dectin-1–induced responses but is not required
for TLR- or TNF-a–triggered activation
Stimulation of Xiap2/2 BMDMs with LPS (for TLR4), Pam3CSK4
(for TLR2), poly (I:C) (for TLR3), or R848 (for TLR7/8) generated
comparable amounts of TNF-a and interleukin (IL)-6 as wild-type
(WT) BMDMs [Figure 1A-C; data not shown for IL-6 stimulated
by poly (I:C) and R848]. We used zymosan depleted to selectively
activate dectin-1 in WT and Xiap2/2 bone marrow-derived dendritic
cells (BMDCs). Dectin-1–induced production of TNF-a, IL-6, and
IL-10 was attenuated in Xiap2/2 BMDCs (Figure 1D-F). Similarly,
XIAP-knockdown human macrophages (Figure 1G) reacted normally to LPS stimulation (supplemental Figure 1A-B) and responded
poorly to dectin-1 stimulation during the production of TNF-a
and IL-6 (Figure 1H-I). XIAP deficiency did not affect the surface
expression of dectin-1 and dectin-2 (supplemental Figure 2A-B).
Dectin-1–induced activation of Syk, extracellular signal-regulated
kinase, and c-Jun N-terminal kinase was comparable between
control and Xiap2/2 macrophages (supplemental Figure 2C-D).
XIAP deficiency specifically reduced dectin-1–induced IkBa
phosphorylation, IkB degradation (supplemental Figure 2E), and
p65 nuclear entry, as well as p38 MAPK activation in macrophages (Figure 1J-L). In contrast, comparable NF-kB activation
was observed in WT and Xiap2/2 macrophages activated by
Pam3CSK4 and LPS (supplemental Figure 3A-B) and in WT and
Xiap2/2 T cells stimulated by TNF-a and LPS (supplemental
Figure 3C-D). Therefore, XIAP is required for dectin-1–induced
NF-kB activation but is not apparently involved in NF-kB activation stimulated by TNF-a, Pam3CSK4, LPS, poly (I:C), and
R848 in T cells and macrophages.
XIAP interacts with BCL10 and promotes BCL10
K63 ubiquitination
Because XIAP participates in dectin-1–induced, but not TNF-a– and
TLR-triggered NF-kB activation, the NF-kB activation stage that
would be regulated by XIAP should be upstream of transforming
growth factor-beta-activated kinase 1-IkB kinase activation. Dectin1–induced NF-kB activation differs from TNF-a– and LPS-triggered
NF-kB activation in the selective formation of the CARD9-BCL10MALT1 complex.23-28 A previous study suggested an association of
XIAP with BCL10.33 Immunoprecipitation of the overexpressed
XIAP-Myc brought down FLAG-BCL10 but not FLAG-MALT1
(supplemental Figure 4). The specific interaction between endogenous XIAP and BCL10 was confirmed in curdlan-stimulated macrophages (Figure 2A). XIAP consists of the N-terminal baculovirus IAP
repeat (BIR) 1, BIR2, BIR3, and C-terminal really interesting new
gene finger domains.12 Using FLAG-tagged XIAP deletion mutants,
we then mapped the domain of XIAP that binds BCL10. The
BCL10-binding region was isolated to the BIR1 segment of XIAP
(Figure 2B).
We examined whether XIAP promotes BCL10 polyubiquitination. Indeed, coexpression with XIAP increased the polyubiquitination of BCL10 in vivo (Figure 2C). However, deletion of the RING
finger domain from XIAP (XIAPDRF) or the removal of BIR1
eliminated the ability of XIAP to ubiquitinate BCL10 (Figure 2C).
Further, the incorporation of K63 Ub into BCL10 was enhanced by
XIAP, whereas the addition of K48 Ub to BCL10 was not affected by
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Figure 1. Defective responses of XIAP-deficient macrophages to dectin-1 agonist but not to LPS Pam3CSK4, poly (I:C), or R848. (A-C) Comparable LPS-, Pam3CSK4-,
R848-, or poly (I:C)-induced TNF-a and IL-6 production in control and Xiap2/2 cells. Control and Xiap2/2 BMDMs were activated with LPS (250 ng/mL), Pam3CSK4 (500 ng/mL),
R848 (2 mg/mL), or poly (I:C) (50 mg/mL), and the secretion of (A,C) TNF-a and (B) IL-6 was quantitated 18 hours later by enzyme-linked immunosorbent assay (ELISA) using IL-6
DuoSet and TNF-a DuoSet. (D-F) XIAP-knockout reduced dectin-1–induced cytokine secretion. Control and Xiap2/2 BMDMs were seeded overnight and treated with zymosan
depleted (100 mg/mL) to activate dectin-1. The (D) TNF-a, (E) IL-6, and (F) IL-10 generated were quantitated 16 hours after activation by ELISA. (G) Knockdown of XIAP in human
macrophages. Human macrophages were differentiated from peripheral monocytes and were transfected with control and XIAP-siRNA. The expression levels of XIAP were
determined 48 hours after transfection. (H-I) Reduced dectin-1–induced TNF-a and IL-6 in XIAP-knockdown human macrophages. Control and XIAP-knockdown human
macrophages (48 hours after transfection) were seeded overnight and treated with zymosan depleted (100 mg/mL) to activate dectin-1. The TNF-a and IL-6 generated were
quantitated 16 hours after activation by ELISA. **P , .01 and ***P , .001 for paired Student t test. Each data point represents mean of triplicate determinations in a single
experiment, and each experiment (A-I) was repeated 3 times with similar results. (J-L) Impaired NF-kB activation and p38 MAPK phosphorylation in Xiap2/2 BMDMs stimulated
with dectin-1 ligands. (J,L) Total cell lysates and (K) nuclear extracts were prepared from BMDMs stimulated with (J-K) zymosan depleted or (L) curdlan at the indicated time points.
(J,L) IkBa and p38 phosphorylation in total cell lysates and (K) the entry of p65 in nuclear extracts were determined by immunoblotting. HSP90 and HDAC2 were the internal
controls for the total cell lysate or nuclear extract, respectively. (J-L) were repeated twice with similar results.
XIAP in 293T cells (supplemental Figure 4B). We also used in vitro
ubiquitination analysis to show that, in reaction mixtures containing
ubiquitin, E1, E2, and recombinant BCL10, the addition of recombinant XIAP, but not XIAPΔRF, induced K63 ubiquitination of
BCL10 (Figure 2D).
We next examined the possible XIAP-mediated ubiquitination
sites on BCL10. Ubiquitination of Lys31 and Lys63 on BCL10 has
been functionally linked to the activation of NF-kB.34 Mutation of
Lys31 and Lys63 on BCL10 (BCL10-KKRR) prevented XIAPmediated K63 ubiquitination (Figure 2E), suggesting that K31 and
K63 on BCL10 are specifically targeted for ubiquitination by XIAP.
In addition, BCL10-stimulated kB-Luc activation35 was profoundly
enhanced by the coexpression of WT XIAP but not by XIAPDBIR1
or XIAPDRF (Figure 2F). Therefore, the association of XIAP with
BCL10 and the ubiquitination of BCL10 modulated the ability of
BCL10 to activate NF-kB.
XIAP deficiency impairs BCL10-mediated NF-kB activation
stimulated by T-cell receptor, lysophosphatidic acid,
and epidermal growth factor receptor
The CARMA1-BCL10 complex is involved in NF-kB activation triggered through the T-cell receptor (TCR) and B-cell
receptor.34,36-38 CARMA3 and BCL10 are required for NF-kB
activation induced by epidermal growth factor (EGF) and G proteincoupled receptor agonists such as lysophosphatidic acid (LPA).39-43
We therefore examined whether XIAP also participated in NF-kB
activation stimulated by these receptors. In the absence of CD28
costimulation, XIAP deficiency led to attenuated CD3-stimulated
interferon (IFN)-g production (Figure 3A), deceased TCR-induced
IKKa/b phosphorylation (Figure 3B), and reduced anti–CD3induced RelA nuclear translocation (Figure 3C). It may be noted
that XIAP deficiency did not affect CD3/CD28-induced IKKa/b
phosphorylation and IL-2 production (supplemental Figure 5),
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Figure 2. XIAP interacts with BCL10 and promotes BCL10 K63 polyubiquitination and BCL10-directed NF-kB activation. (A) Interaction between endogenous XIAP
and BCL10 in macrophages. BMDMs were activated with curdlan, and total cell lysates were prepared at the indicated time points. Total cell lysates (1 mg) were
immunoprecipitated with anti-BCL10, and the presence of XIAP, MALT1, and BCL10 was determined. (B) BCL10 interacts with the BIR1 domain of XIAP. FLAG-tagged fulllength (FL), deleted RING finger (DR), N-terminal (N), C-terminal (C), BIR1, BIR2, or BIR3 of XIAP were cotransfected with BCL10-Myc into 293T. Total cell lysates were
immunoprecipitated by anti-Myc, and the presence of XIAP and its mutants in immune complexes was detected by anti-FLAG. (C) FL-XIAP, but neither XIAPDRF nor
XIAPDBIR1, promotes BCL10 polyubiquitination. HA-ubiquitin, BCL10, XIAP, XIAPDRF, or XIAPDBIR1 (DB) was transfected into 293T cells as indicated. Total cell lysates
were prepared and immunoprecipitated by anti-BCL10, and Ub-containing protein was detected by anti-HA. (D) BCL10 K63 ubiquitination was induced by XIAP in vitro. In vitro
ubiquitination assays were conducted in reaction mixtures containing human ubiquitin-K63, human E1, E2 (Ubc13), BCL10-FLAG, XIAP, or XIAPDRF, as indicated. BCL10FLAG was loaded on protein G magnetic beads through anti-FLAG. Reactions proceeded at 30°C for 1 hour. BCL10-FLAG-protein G was pulled down, separated on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted with ubiquitin antibody (upper panel). The input is shown in the lower panel. (E) Lys31 and Lys63 on BCL10
were required for XIAP-mediated K63 ubiquitination on BCL10. WT-BCL10 or BCL10-KKRR (K31R, K63R) was cotransfected with XIAP and K63 Ub into 293T cells. Total cell
lysates were immunoprecipitated by anti-BCL10. Ubiquitinated BCL10 of the immune complexes was detected by anti-HA antibody. (F) XIAP enhances BCL10-activated kB
reporter. kB-Luc was transfected with EGFP, XIAP, XIAPDRF, XIAPDBIR1, and BCL10, as indicated, into 293T cells, and luciferase activity was determined 24 hours later.
EGFP was used to determine transfection efficiency. ***P , .001 for paired Student t test. (A-F) Each experiment was repeated 3 times with similar results.
consistent with the effect previously reported in Xiap2/2 T cells13
and CARMA1-knockout T cells.44
We also used Xiap2/2 macrophages to confirm that LPA-triggered
IL-6 production and p65 nuclear translocation were decreased in
Xiap2/2 macrophages (Figure 3D-E). In addition, EGF-stimulated
IkBa phosphorylation and p65 nuclear entry was reduced in XIAPknockdown HepG2 cells (Figure 3F-G). Therefore, consistent with
the suggestion that XIAP is involved in BCL10-mediated NF-kB
signaling, XIAP is required for full NF-kB activation triggered by
dectin-1, LPA, epidermal growth factor receptor (EGFR), or TCR
alone.
Sensitivity of Xiap2/2 mouse to C albicans infection
C albicans is recognized by dectin-1 and dectin-2, and the fungal
infection is controlled by dectin-induced responses through the
CARD9-BCL10-NF-kB pathway.23-27,45,46 We therefore tested
whether deficiency in XIAP leads to defective responses toward
C albicans. We found that 8- to 10-week-old Xiap2/2 mice were highly
susceptible to C albicans (1 3 106) infection, with all mice dying
from infection by day 3 (Figure 4A). Older Xiap2/2 mice (16 weeks
old) were modestly more resistant to C albicans (1 3 106; Figure 4B).
Immediately after C albicans administration, generation of IL-6,
monocyte chemoattractant protein (MCP)-1, and TNF-a was diminished in Xiap2/2 mice relative to WT mice (Figure 4C). At a lower
dose of C albicans (1 3 105), Xiap1/1 mice were fully resistant,
whereas most of the Xiap2/2 mice died from infection before day 15
(Figure 4D), accompanied by progressive body weight loss. In these
dying Xiap2/2 mice, scattered foci of abscesses were observed in the
swollen kidneys (Figure 4E), as well as a large fraction of infiltrated
neutrophils in kidney (Figure 4F). C albicans titers were also highly
elevated in the kidney, liver, and spleen tissues of Xiap2/2 mice
(Figure 4G). The compromised responses to C albicans in XIAPdeficient mice were comparable to those seen in mice lacking dectin-1,
dectin-2, or CARD9.23,28,45,46 Another consequence of XIAP deficiency was found in the accumulation of proinflammatory cytokines
following a low dose of C albicans infection. Although the serum
contents of IL-6, TNF-a, and MCP-1 were higher in WT mice than
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Figure 3. XIAP deficiency impairs TCR-, LPA-, and EGF-stimulated cytokine production and NF-kB activation. (A) Diminished IFN-g production in Xiap2/2 T cells.
Purified T cells from normal littermate control and Xiap2/2 mice were activated with plate-bound anti-CD3 (5 mg/mL), and the IFN-g secretion was determined 48 hours later.
(B-C) Reduced CD3-induced NF-kB activation in Xiap2/2 T cells. Control and Xiap2/2 T cells were activated with anti-CD3, and (B) total cell lysates and (C) nuclear extracts
were prepared at the indicated time points. (B) IKK phosphorylation in total cell lysates and (C) the presence of p65 in nuclear extracts were determined by immunoblotting.
HDAC2 was the internal control of the nuclear extract, whereas glyceraldehyde-3-phosphate dehydrogenase was the marker of the cytosolic extract. (D-E) Diminished LPAstimulated IL-6 production and NF-kB activation in Xiap-deficient macrophages. Peritoneal macrophages were isolated from WT and Xiap2/2 mice primed with thioglycolate
and were stimulated with LPA (10 mM). (E) Nuclear extracts were prepared at 30 and 60 minutes, and the presence of p65 as determined. (D) IL-6 was quantitated by ELISA
16 hours later. (F-G) XIAP knockdown decreased EGF-induced NF-kB activation. Control siRNA or XIAP-specific siRNA was transfected into HepG2 cells by electroporation.
HepG2 cells were plated 16 hours after transfection and treated with EGF (20 ng/mL) after another 24 hours. Total cell lysates and nuclear extracts were prepared at the
indicated time points. (F) EGF-induced IkBa phosphorylation and (G) p65 nuclear translocation were measured in total cell lysates and nuclear extracts,
respectively. *P , .05, **P , .01, and ***P , .001 for paired Student t test. (A,D) Each data point represents mean of triplicate determinations in a single experiment, and
(A-G) each experiment was repeated 3 times with similar results.
Xiap2/2 mice 6 hours after a low dose of C albicans infection (data
not shown), these cytokines were no longer detectable in the peripheral
blood of WT mice 1 week after infection (Figure 4H), consistent
with the near clearance of C albicans in these mice (Figure 4G). In
contrast, Xiap2/2 mice retained a high serum level of IL-6, TNF-a,
and MCP-1 at the same time point (Figure 4H), suggesting that the continued presence of C albicans turns the defective innate responses
into a proinflammatory status late in infection. A correlation was also
found between the extent of the body weight loss and serum levels
of IL-6, TNF-a, and MCP-1 in those Xiap2/2 mice 1 week after
C albicans infection (supplemental Figure 6), illustrating that high levels
of proinflammatory cytokines are associated with poor prognosis.
We also measured T-cell responses to HKCA in WT and Xiap2/2
mice. After immunization with HKCA, the production of IFN-g and
IL-4 was comparable between WT and Xiap2/2 T cells (supplemental Figure 7A-B). Because T-cell response was not affected
as obviously as macrophages in Xiap2/2 mice, we focused on the
dectin-1–induced innate immunity in Xiap2/2 mice during the rest of
study.
Dectin-1 ligand curdlan induces XLP-2–like syndromes in
Xiap2/2 mouse
To further delineate the effect of XIAP in the BCL10-dependent
innate response, we used a model dectin-1 ligand, curdlan. Although
WT mice did not display any abnormalities following weekly curdlan
intraperitoneal immunization, Xiap2/2 mice exhibited body mass
loss 1 week after curdlan priming (Figure 5A) and did not recover
until the fourth week (data not shown). Serum levels of IL-6, IL-10,
MCP-1, and TNF-a were decreased in Xiap2/2 mice after administration of curdlan (Figure 5B). Curdlan-induced immediate peritoneal infiltration of macrophages and neutrophils was reduced in
Xiap2/2 mice (Figure 5C). The compromised early innate responses
to curdlan in Xiap2/2 mice were reversed a week later. The generation
of IL-6, IL-10, TNF-a, and MCP-1 were much higher in Xiap2/2
mice than the WT control (Figure 5D). Splenomegaly was found
in Xiap2/2 mice 1 week after curdlan priming, with profound
infiltration of mononuclear cells (Figure 5E). Also, the peritoneal
presence of macrophages and neutrophils became higher in
Xiap2/2 mice than in control mice 1 week after curdlan administration (Figure 5F).
As a control, immunization of Pam3CSK4 did not generate any
differential response between control and Xiap2/2 mice (supplemental Figure 8). Repetitive administration of Pam3CSK4 did not
produce detrimental effects in Xiap2/2 mice (data not shown).
Because Xiap2/2 mice reacted normally to Pam3CSK4 in NF-kB
activation and inflammatory cytokine production (Figure 1A-B;
supplemental Figure 3A), we examined whether priming of
Pam3CSK4 increased the resistance of Xiap2/2 mice to C albicans
infection. Prior administration of Pam3CSK4 did not rescue Xiap2/2
mice from C albicans (1 3 105) infection, despite an increase
in the proinflammatory cytokine levels (supplemental Figure 9A-C).
Instead, Pam3CSK4 accelerated C albicans-mediated mortality
in both control and Xiap2/2 mice (supplemental Figure 9D-E),
indicating that restoration of BCL10-dependent NF-kB activation
and inflammatory cytokine expression in Xiap2/2 mice failed to
compensate for the defective innate response to C albicans.
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Figure 4. Xiap2/2 mice exhibit heightened sensitivity to C albicans infection. (A-B) Poor survival of Xiap2/2 mice following C albicans infection. C albicans (1 3 106) was
intravenously administered to (A) 8- to 10-week-old and (B) 16-week-old WT and Xiap2/2 mice. Survival of mice is presented by a Kaplan-Meier survival curve; n 5 7 for A and B.
(C) Reduced inflammatory cytokine production in Xiap2/2 mice after C albicans infection. Serum from mice in A and B was collected 4 hours after C albicans injection, and
the levels of IL-6, MCP-1, and TNF-a were determined; n 5 10, 3 mice from A and 7 mice from B. (D) Poor survival of Xiap2/2 mice following a low dose of C albicans
infection. WT and Xiap2/2 mice (8-12 weeks old) were intravenously injected with C albicans (1 3 105), and the survival of mice was monitored; n 5 6. (E) Morphology of
kidney from WT and Xiap2/2 mice infected with low doses of C albicans. WT and Xiap2/2 mice were intravenously injected with C albicans (1 3 105). Infected Xiap2/2 mice
were euthanized on 30% loss of body weight. Kidneys were isolated from Xiap2/2 mice and WT mice infected at the same time (days 8-12 after infection). Two pairs of kidney
(of 5) are shown. (F) Elevated neutrophil infiltration in C albicans-infected Xiap2/2 mice kidney. Total kidney cells were prepared from the kidneys in E and stained with antiF4/80 and anti-Gr-1, and the fraction of the macrophages (Gr-12/intF4/801) and neutrophils (Gr-11F4/802) was analyzed by flow cytometry. (G) Highly increased fungal
burden in Xiap2/2 mice infected with C albicans. Livers, kidneys, and spleens were removed from C albicans-infected WT and Xiap2/2 mice in D between days 8 and 12.
Colony-forming units of C albicans in each organ was determined. (H) Elevated inflammatory cytokine in Xiap2/2 mice 1 week after C albicans infection. Serum from mice in D
was collected 7 days after C albicans (1 3 105) injection, and the levels of IL-6, TNF-a, and MCP-1 were determined. *P , .05, **P , .01, and ***P , .001 for paired Student t test.
Impaired dectin-1–mediated Rac1 activation and phagocytosis
in XIAP-deficient macrophages
BCL10 was recently shown to participate in Rac1 activation, F-actin
remodeling, and phagosome closure that are independent of NF-kB
activation.47 We found that curdlan-induced Rac1 activation was
attenuated in Xiap2/2 BMDMs (supplemental Figure 10A). Similarly, dectin-1–induced Rac1 activation was impaired in XIAPknockdown human macrophages (Figure 6A). For phagocytosis, the
uptake of plain latex beads or Pam3CSK4-coated latex in Xiap2/2
BMDMs was not affected, as measured by confocal microscopy
or flow cytometry (supplemental Figure 10B-C). In contrast,
the phagocytosis of HKCA was impaired in Xiap 2/2 BMDMs
(supplemental Figure 10D). Similar to Xiap2/2 BMDMs, XIAPknockdown human macrophages were defective in the phagocytosis of HKCA (Figure 6B). Quantitation by flow cytometry further
confirmed the impaired uptake of C albicans in XIAP-deficient
human macrophages (Figure 6C). We also introduced active Rac1
(Rac1-V12) into XIAP-knockdown human macrophages, shown
by the constitutive Rac1 activity (Figure 6A). The defective uptake
of C albicans was reversed by the expression of Rac1-V12 in
XIAP-knockdown human macrophages (Figure 6B-C), suggesting
that impaired Rac1 activation accounts for the ineffective uptake of
HKCA in XIAP-deficient macrophages.
We then looked for reagents that may correct the defect of
Xiap2/2 macrophages. Resolvin D1 is an anti-inflammatory lipid
mediator biosynthesized from v-3 fatty acid docosahexaenoic
acid.30,31 Resolvin D1 displays a specific activity to enhance phagocytosis in macrophages.32,48-50 We found that aspirin-triggered
resolvin D1 (AT-RvD1) restored dectin-1–induced Rac1 activation
in Xiap2/2 BMDMs (supplemental Figure 11A). RvD1 did not
further increase the phagocytosis of HKCA in control BMDMs but
significantly increased the uptake of HKCA in Xiap2/2 BMDMs
(supplemental Figure 11B-C). A similar effect of RvD1 was
found in XIAP-knockdown human macrophages. The addition
of RvD1 corrected the defective dectin-1–induced Rac1 activation (Figure 6D) and restored the phagocytosis of HKCA in
XIAP-deficient human macrophages (Figure 6E-F). Notably,
RvD1 did not increase dectin-1–mediated p38 MAPK phosphorylation or NF-kB activation in Xiap2/2 BMDMs (supplemental
Figure 11D), indicating that the effect of RvD1 is independent of
p38 and NF-kB.
Resolution of inflammation rescues Xiap2/2 mouse from
C albicans infection
We then examined the in vivo effect of RvD1 in Xiap2/2 mice.
Administration of RvD1 3 days after C albicans (1 3 105) infection
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Figure 5. Xiap-deficient mice were unable to resolve the presence of curdlan. (A) Loss of body weight in Xiap 2 /2 mice with weekly treatment of curdlan. Female
mice (8-12 weeks old) were primed with intraperitoneal administration of curdlan (5 mg/20 g body weight) every week, and body weight was determined; n 5 13.
(B) Attenuated inflammatory cytokine production in the Xiap 2 /2 mouse immediately after curdlan immunization. Serum was collected 2 hours after curdlan
priming, and the quantity of IL-6, IL-12, MCP-1, and TNF-a was determined; n 5 5. (C) Reduced recruitment of macrophages and neutrophils in the Xiap 2 /2
mouse. Peritoneal cavity infiltrates were collected 4 hours after curdlan priming, cell numbers were counted, and the population of macrophages and neutrophils
was analyzed by flow cytometry. (D) Enhanced inflammatory cytokines secretion in Xiap 2 /2 mice 1 week after curdlan stimulation. Serum was collected from mice
1 week after immunization of curdlan, and the contents of cytokines were quantitated; n 5 5. (E) Curdlan treatment leads to splenomegaly in Xiap 2 /2 mice.
Spleens were isolated from control and Xiap 2 /2 mice 7 days after curdlan immunization for comparison. Sections of spleen were stained with hematoxylin and
eosin. One representative pair (of 5 pairs) is shown. Bar indicates 50 mm. (F) Increased macrophage and neutrophil infiltration in Xiap 2 /2 mice 1 week after
curdlan stimulation. Cells were harvested from the peritoneal cavity in mice from D, and the quantities of macrophages and neutrophils were determined. *P , .05,
**P , .01, and ***P , .001 for paired Student t test.
fully rescued Xiap2/2 mice from C albicans-mediated mortality
(Figure 7A). Development of the pathological morphology in the
kidneys of Xiap2/2 mice after C albicans infection was completely
prevented by RvD1 (Figure 7B). For reasons that are unclear,
kidneys from 4 of the 6 RvD1-treated Xiap2/2 mice appeared to
be smaller than those from RvD1-treated WT mice. Neutrophil
infiltration in the kidney from C albicans-infected Xiap2/2 mice
was abrogated by RvD1 administration (Figure 7C). The high
levels of IL-6, TNF-a, and MCP-1 in Xiap2/2 mice 10 days after
C albicans infection was no longer detectable after RvD1 administration (Figure 7D-F). The fungal loads in liver, spleen, and
kidney were effectively reduced by RvD1 treatment (Figure 7I-K).
RvD1 also increases the production of IL-10,49,51 known to participate
in the resolution of inflammation. We found that curdlan-triggered
IL-10 generation in Xiap2/2 BMDCs was not altered by the presence of AT-RvD1 (Figure 7G). Application of RvD1 did not increase
serum levels of IL-10 in Xiap2/2 mice with prior C albicans infection
(Figure 7H). Together, these results suggest that increases in
dectin-1–induced Rac1 activation and phagocytosis by RvD1
helped Xiap2/2 mice resolve inflammation and survive C albicans
infection.
Discussion
XIAP is a caspase-binding antiapoptotic protein critical for cell
survival and has been shown to activate NF-kB. XIAP deficiency in
humans leads to XLP-2, although the mechanisms remain unclear.
However XIAP-knockout mice appear normal.13-15 In the present
study, we identified the involvement of XIAP in selective NF-kB
activation. We showed that TNF-a/IL-6 production induced by
TLR2, TLR3, TLR4, or TLR7/8 was normal in Xiap2/2 BMDMs,
and XIAP deficiency did not affect TLR-induced NF-kB activation in T cells and macrophages, consistent with several recent
reports.16,20-22 XIAP deficiency impaired only NF-kB activating
processes that are mediated by BCL10, such as those stimulated by
dectin-1, TCR, EGF, or LPA (Figures 1 and 3). Our data also agree
with the observations that TNF-a–triggered NF-kB activation is
normal in BCL10-knockout cells40 and that LPS-induced NF-kB
activation is not affected by deficiency in CARD9,23 the BCL10binding partner. We demonstrated that XIAP promoted BCL10
K63 ubiquitination, but further work is required to delineate the
exact role of XIAP-BCL10 in CARMA1-BCL10-MALT1–mediated
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HSIEH et al
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
Figure 6. Defective dectin-1–mediated Rac1 activation and phagocytosis in XIAP-deficient human macrophages are corrected by resolvin D1. (A) Impaired Rac1
activity in XIAP-knockout human macrophages. Control (Ctrl) and XIAP-knockdown (small interfering XIAP) human macrophages, with or without active
Rac1 (Rac1V12) transfected, were incubated with curdlan (100 mg/mL) and harvested at 0 and 10 minutes. The activation of Rac1 was measured by the G-LISA
Rac1 activation assay kit. (B-C) Defective phagocytosis of C albicans in XIAP-knockdown human macrophages and restoration by Rac1V12 expression. Control and
XIAP-knockdown human macrophages, with or without Rac1V12 transfected, were incubated with fluorescein isothiocyanate-conjugated heat killed C albicans
(FITC-HKCA) for 30 minutes. Cells were collected at 0 and 30 minutes and analyzed by (B) confocal microscopy and (C) flow cytometry. (B) Cells were fixed,
permeabilized, and stained with Alexa Fluor 594-Phalloidin (for F-actin) and 49,6 diamidino-2-phenylindole for confocal microscopy. Bar indicates 20 mm. (C) Cells
were also treated with trypan blue to quench the extracellular associated FITC, and the internalized FITC-conjugated bead was determined by flow cytometry. Gray
shadow, fluorescence-activated cell sorting (FACS) profile at 0 minutes; blue and red curves, uptake of FITC-HKCA at 30 minutes; orange curve, internalization of
FITC-HKCA at 30 minutes by Rac1V12-transfected macrophages. (D) Resolvin D1 restores Rac1 activity in XIAP-deficient human macrophages. Control and XIAPknockdown human macrophages were incubated with curdlan (100 mg/mL) with or without AT-resolvin D1 (RvD1, 100 nM) pretreatment. Cells were harvested at
0 and 10 minutes, and the activation of Rac1 was measured. (E-F) Resolvin D1 restores phagocytosis of C albicans in XIAP-knockdown human macrophages.
Control and XIAP-knockdown human macrophages, pretreated with vehicle or RvD1 (100 nM), were incubated with FITC-HKCA for 30 minutes. Cells were collected
at 0 and 30 minutes and analyzed by (E) confocal microscopy and (F) flow cytometry. Gray shadowed curve, FACS profile at 0 minutes; blue and red curves,
internalization of FITC-HKCA at 30 minutes; green curve, internalization of FITC-HKCA at 30 minutes by RvD1-treated macrophages. *P , .05 and **P , .01 for
paired Student t test. Confocal images were obtained a Zeiss LSM 780 confocal microscope (Jena, Germany) with an objective lens of plan-Apochromat 633/1.4 Oil
DIC M27 at room temperature. 49,6 Diamidino-2-phenylindole fluorescence was excited at 405 nm and collected at 420 to 470 nm. FITC fluorescence was excited at
488 nm and collected at 500 to 550 nm. Cy3 fluorescence (transfection indicator, not shown in figure) was excited at 561 nm and collected at 568 to 629 nm. Alexa
Fluor 594 fluorescence was excited at 633 nm and collected at 641 to 758 nm. The pinhole was at 61.7 mm, with an 11.4-mm section. Images were taken by an
AxioCam digital microscope camera (Zeiss) using Carl Zeiss software Zen 2010B SP1, version 6.0.
NF-kB activation. TCR-stimulated NF-kB in Xiap2/2 T cells was
indistinguishable from control T cells when stimulated through
CD3/CD28 (supplemental Figure 5). A defect in T-cell activation
was observed in Xiap2/2 T cells only when activated by anti-CD3
alone (Figure 3A). We also found that immunization with HKCA
generated comparable T-cell responses in WT and Xiap2/2 mice
(supplemental Figure 7). Despite the fact that XIAP targets to
BCL10, there was a different sensitivity to TCR and dectin-1 stimulation in Xiap2/2 T cells and macrophages. Further work will be
needed to delineate this discrepancy.
We demonstrated that Xiap2/2 mice died from C albicans infection. XLP-2 is known for its association with Epstein-Barr virus,
and no C albicans infection has been reported in XLP-2, likely due
to the rarity of XLP-2 patients. Because Xiap2/2 T-cell responses
are nearly normal, XLP-2 may be attributed to abnormal innate
immunity in XIAP-deficient hosts. We further illustrated that the
defective BCL10-dependent uptake of C albicans plays a major
role in the vulnerability of Xiap2/2 mice to C albicans infection.
BCL10 participates in Rac1 activation, F-actin remodeling, and
phagosome formation that are NF-kB independent.47 XIAP-deficient
macrophages displayed no defects in the phagocytosis of plain latex
beads and Pam3CSK4-coated latex beads but were ineffective in
the uptake of C albicans, indicating a selective deficiency in dectin1–induced phagocytosis. Introduction of active Rac1 restored the
phagocytosis of C albicans in XIAP-deficient macrophages
(Figure 6A-C). Up-regulation in NF-kB activation and proinflammatory cytokine production by Pam3CSK4 failed to rescue Xiap2/2
mice from C albicans-mediated inflammation and lethality
(supplemental Figure 9). Instead, restoration in the Rac1 activation
and uptake of C albicans effectively led to clearance of infection
(Figure 7). The inability of Xiap2/2 mice to respond to C albicans
infection thus could be attributed mostly to the impaired BCL10dependent Rac1 activation and phagocytosis necessary for yeast
clearance.
Importantly, we demonstrated that XIAP-deficient human macrophages recapitulated the specific defects in Xiap2/2 mouse macrophages (Figures 1 and 6). XIAP-knockout human macrophages
reacted indistinguishably from control macrophages to LPS
treatment (supplemental Figure 1) but were unable to respond to
dectin-1 stimulation (Figure 1H-I). Similar to Xiap2/2 macrophages,
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Figure 7. Resolvin D1 effectively rescues Xiap2/2 mice from C albicans infection. (A) Resolvin D1 rescues Xiap2/2 mice from C albicans infection. C albicans (1 3 105)
was intravenously administered to Xiap2/2 mice (8-12 weeks old), with or without intravenous injection of RvD1 (100 ng)30-32 3 days later. The survival of mice was monitored
and is presented as a Kaplan-Meier survival curve; n 5 6. (B) Resolvin D1 restores the normal morphology of kidney from C albicans-infected Xiap2/2 mice. WT (Ctrl) and
Xiap2/2 mice were treated as in A. Infected Xiap2/2 mice were euthanized on 30% loss of body weight. Kidneys were isolated from Xiap2/2 mice and WT mice infected at the
same time. Kidneys shown were representative of 6 mice. (C) Resolvin D1 alleviates kidney neutrophil infiltration in C albicans-infected Xiap2/2 mice. Total kidney cells were
prepared from kidneys isolated in B, and the fraction of neutrophils was determined. (D-F,H) Resolvin D1 resolves the persistent inflammatory cytokine levels in Xiap2/2 mice
after C albicans infection. Serum from C albicans-infected mice was collected 6 hours and 10 days after infection, and the levels of (D) IL-6, (E) TNF-a, (F) MCP-1, and (H)
IL-10 were determined. (G) Resolvin D1 does not increase IL-10 production. WT and Xiap2/2 BMDCs were pretreated with vehicle or resolvin D1 and stimulated with curdlan.
Secreted IL-10 was determined 24 hours after activation. (I-K) Resolvin D1 eliminates the fungal burden in Xiap2/2 mice infected with C albicans. (I) Livers, (J) kidneys, and
(K) spleens were removed form C albicans-infected WT and Xiap2/2 mice in day 10. Colony-forming units of C. albicans in each organ was determined. *P , .05, **P , .01,
and ***P , .001 for paired Student t test.
XIAP-deficient human macrophages were compromised in
dectin-1–stimulated Rac1 activation and were deficient in the
uptake of C albicans. The present study likely represents the first
identification of the physiological defect in XLP-2 macrophages. XLP-2 patients are characterized by hemophagocytic
lymphohistiocytosis. 9,10 Whether impaired Rac1 activation
and defective phagocytosis lead to visible remains of erythrocytes and leukocytes in XLP-2 macrophages remains to be
determined.
We further used the dectin-1 ligand curdlan to demonstrate that
the reduced innate responses in Xiap2/2 mice lead to development of
selected XLP-2 syndromes. The initial inability of Xiap2/2 mice to
mount effective innate immunity to curdlan (Figure 5B) resulted in
the persistent presence of curdlan, and its continued stimulation was
accompanied by accumulation of the proinflammatory cytokines,
leading to elevated accumulation of macrophages and neutrophils in
the peritoneal cavity and splenomegaly (Figure 5D-F). Therefore, the
inability of Xiap2/2 mice to generate effective immune responses to
curdlan and C albicans results in some inflammatory syndromes seen
in XLP-2 patients.
It has been shown that moderate T-cell immunodeficiency, but not
severe T-cell immunodeficiency, generates immune dysregulation and
autoimmunity.52 We propose that XIAP deficiency is an example
that partial innate immunodeficiency generates uncontrolled
inflammatory consequences. In an SPF environment without
pathogenic challenge, Xiap2/2 mice are indistinguishable from
WT mice. However, for microbes or pathogen-associated molecular patterns that activate BCL10-dependent pattern recognition receptors, Xiap2/2 mice are unable to mount effective innate
immune responses to resolve infection or eliminate pathogenassociated molecular patterns, and their continued stimulation leads
to inflammation and XLP-2–like syndromes including persistent
activation of macrophages, excess production of inflammatory
cytokines, and splenomegaly. Our results suggest that defective
BCL10/NOD2-directed innate activation represents one of the
mechanisms that contribute to the pathogenesis of XLP-2 in XIAPdeficient patients. Because XIAP deficiency impairs the innate
reactivity of myeloid cells, our results may explain why myeloablative conditioning regimens lead to poor survival in allogeneic
hematopoietic cell transplantation on XIAP-mutated patients,53 as
myeloid cell depletion further aggravates pathogenesis caused by
innate immunodeficiency.
Innate immunodeficiency is characterized by sensitivity to a
narrow spectrum of infection.1-3 Increasing numbers of inflammatory
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HSIEH et al
diseases, including cystic fibrosis, are attributed to innate immunodeficiency.54 In the present study, we used Xiap2/2 mice to suggest a
molecular basis of such selectivity. An interesting analogy is that
Crohn disease-associated NOD2 variants are loss-of-function
mutants.55 How the loss of function of a proinflammatory signal
molecule mechanistically contributes to an inflammatory disorder
has been subjected to extensive investigations.5-7 It has been
suggested that patients with NOD2 mutants are unable to resolve
infection by specific pathogens due to innate immunodeficiency.5,7
It is likely that the persistence of microbes leads to continued
inflammation and adaptive immunity activation, similar to what we
observed in this study in Xiap2/2 mice.
We also demonstrated that RvD1 restored Rac1 activation and
phagocytosis in XIAP-deficient macrophages, resulting in effective
resolution of inflammation, elimination of C albicans, and full
survival of the infected Xiap2/2 mice. Interestingly, RvD1 did not
rescue the activation of NF-kB and p38 in Xiap2/2 macrophages.
These results suggest that, for innate immunodeficiency-induced inflammatory syndromes such as XLP-2, after the initial failure of
host responses to clear pathogen, resolution of the latter-stage
inflammation is likely the key for the success in the therapy.
Furthermore, our results suggest that increased uptake of pathogens
and resolution of the inflammation by RvD1 are potential therapeutic
interventions for inflammatory diseases caused by primary innate
immunodeficiency. Further work will determine the feasibility of
such an approach.
Acknowledgments
The authors thank Dr David L. Vaux for the Xiap2/2 mouse and
Yamin Lin and the FACS Core, Sue-Ping Lee, and the Confocal Core
of the Institute of Molecular Biology, Academia Sinica, for cell
sorting and confocal microscopy. Dr AndreAna Peña, English editor
of Institute of Molecular Biology, Academia Sinica, provided editorial
assistance to the authors during preparation of this manuscript.
This work was supported by National Science Council grant 1022321-B-001-045 and an Academia Sinica Investigator Award from
Academia Sinica, Taiwan, Republic of China.
Authorship
Contribution: W.-C.H., Y.-T.C., and I.-H.C. performed experiments;
S.-C.H. and S.-C.M. contributed to generation of key materials and
constructs; and M.-Z.L. conceived of, designed, and supervised the
research and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Ming-Zong Lai, Institute of Molecular Biology,
Academia Sinica, Taipei 11529, Taiwan, Republic of China; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2014 124: 2847-2857
doi:10.1182/blood-2014-03-564609 originally published
online September 4, 2014
Inability to resolve specific infection generates innate immunodeficiency
syndrome in Xiap−/− mice
Wan-Chen Hsieh, Ya-Ting Chuang, I-Hsuan Chiang, Shu-Ching Hsu, Shi-Chuen Miaw and
Ming-Zong Lai
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