Download Increased Susceptibility to Salmonella Infection in Signal Regulatory

Increased Susceptibility to Salmonella
Infection in Signal Regulatory Protein α
-Deficient Mice
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of June 17, 2017.
Lin-Xi Li, Shaikh M. Atif, Shirdi E. Schmiel, Seung-Joo Lee
and Stephen J. McSorley
J Immunol 2012; 189:2537-2544; Prepublished online 30
July 2012;
doi: 10.4049/jimmunol.1200429
http://www.jimmunol.org/content/189/5/2537
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References
The Journal of Immunology
Increased Susceptibility to Salmonella Infection in Signal
Regulatory Protein a-Deficient Mice
Lin-Xi Li,* Shaikh M. Atif,* Shirdi E. Schmiel,† Seung-Joo Lee,* and Stephen J. McSorley*
C
hronic bacterial infections initiate both innate and adaptive immune responses in the host, primarily within the
secondary lymphoid organs, including spleen and lymph
nodes (1, 2). Systemic infection of Salmonella enterica, a gramnegative intracellular bacterium, leads to a characteristic pathological condition called splenomegaly in both mice and humans
(3, 4). At the peak of infection, the spleen of infected C57BL/6
mice expands to .10-fold its size in the naive state (5). It was
commonly thought that the enlarged spleen accompanying Salmonella infection is caused by local leukocyte expansion or recruitment. Indeed, splenic lymphocytes, especially CD4 and CD8
T cells, expand after Salmonella infection, and a large increase in
phagocyte populations is also observed (5–7). Although wild-type
(WT) C57BL/6 mice typically survive and exhibit a self-limiting
infection with an attenuated S. typhimurium strain, immunodeficient mice lacking CD4, MHC class II, IFN-g, or T-bet fail to
clear a primary Salmonella infection, demonstrating an indispensable role for CD4 Th1 cells in Salmonella protective immunity (8–10). In addition, Ab responses are also known to contribute to resistance to Salmonella infection (11–13), although the
mechanism of protection is unclear.
More recently, our laboratory reported that RBCs (defined as
CD712Ter119+) and reticulocyte precursors (CD71+Ter119+) expand greatly during Salmonella infection, increasing from ∼20%
of naive splenocytes to .80% of the infected spleen (5). Given
this large erythroid expansion, these observations provide an alternative mechanism to explain Salmonella-induced splenomeg-
aly. Importantly, blocking erythropoiesis induced by elevated
levels of the hormone erythropoietin (EPO) significantly reduced
host susceptibility to Salmonella infection (5, 14). These data
suggest that increased erythropoiesis is an evasive mechanism that
Salmonella adopts to facilitate persistent infection of the host.
In the steady state, erythropoiesis in the bone marrow and RBC
turnover in the periphery are tightly regulated (15, 16). An important
signaling module that regulates RBC turnover is the SIRPa-CD47
axis. SIRPa is a transmembrane glycoprotein also known as SHPS1 (Src homology 2 domain-containing protein tyrosine phosphatase
substrate-1), CD172a, BIT (brain Ig–like molecule with a tyrosinebased activation motif), MFR (macrophage fusion receptor), or p84
(17–21). It is expressed primarily on myeloid cells, such as macrophages, granulocytes, and myeloid dendritic cells (DCs), but is
barely detectable on B or T lymphocytes (22–26). The extracellular
domain of SIRPa comprised three Ig-like domains, which interact
with its ligand CD47, another member of the Ig superfamily known
as a marker of self on RBCs (24, 27, 28). The binding of CD47 on
RBCs with SIRPa on macrophages delivers an inhibitory “do not
eat me” signal that prevents unwanted phagocytosis and maintains
the peripheral RBC pool (27–31).
Although more recent studies have implied that SIRPa plays an
important role in regulating the homeostasis of T cells, NK cells,
and DCs (32–34), little is known regarding the function of SIRPa
in bacterial infection and the development of adaptive immunity.
In this current study, we have examined the immune response to
Salmonella infection in SIRPa-deficient mice, and report an indispensable role for SIRPa in resolving Salmonella primary infection and establishing an effective Salmonella-specific adaptive
immune response.
*Department of Anatomy, Physiology and Cell Biology, Center for Comparative
Medicine, School of Veterinary Medicine, University of California, Davis, Davis,
CA 95616; and †Microbiology, Immunology, and Cancer Biology Program, University of Minnesota, Minneapolis, MN 55455
Materials and Methods
Received for publication February 3, 2012. Accepted for publication July 3, 2012.
Mice
This work was supported by National Institutes of Health Grants R01AI055743,
R01AI073672, and R21HL112685.
C57BL/6 (B6) mice were purchased from the National Cancer Institute
(Frederick, MD) and The Jackson Laboratory (Bar Harbor, ME). Congenic
Sirpa2/2 mice (B6.129P2-Sirpa, tm1Nog ./Rbrc) were provided by the
RIKEN BioResource Center through the National BioResource Project of
the Ministry of Education, Culture, Sports, Science and Technology, Japan.
These mice had been backcrossed to C57BL/6 for .10 generations. Rag2/2
mice were kindly provided by Dr. D. Masopust (University of Minnesota,
Minneapolis, MN). CD90.1 or CD45.1 congenic, RAG-deficient SM1 TCR
transgenic mice have been previously described (35, 36). Briefly, the SM1
Address correspondence and reprint requests to Dr. Lin-Xi Li, University of California,
Davis, County Road 98 and Hutchison Drive, Davis, CA 95616. E-mail address:
[email protected]
Abbreviations used in this article: DC, dendritic cell; EPO, erythropoietin; HK, heatkilled; WT, wild-type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200429
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Recent studies have shed light on the connection between elevated erythropoetin production/spleen erythropoiesis and increased
susceptibility to Salmonella infection. In this article, we provide another mouse model, the SIRPa-deficient (Sirpa2/2) mouse, that
manifests increased erythropoiesis as well as heightened susceptibility to Salmonella infection. Sirpa2/2 mice succumbed to
systemic infection with attenuated Salmonella, possessing significantly higher bacterial loads in both the spleen and the liver.
Moreover, Salmonella-specific Ab production and Ag-specific CD4 T cells were reduced in Sirpa2/2 mice compared with wild-type
controls. To further characterize the potential mechanism underlying SIRPa-dependent Ag-specific CD4 T cell priming, we
demonstrate that lack of SIRPa expression on dendritic cells results in less efficient Ag processing and presentation in vitro.
Collectively, these findings demonstrate an indispensable role of SIRPa for protective immunity to Salmonella infection. The
Journal of Immunology, 2012, 189: 2537–2544.
2538
ROLE OF SIRPa IN SALMONELLA INFECTION
TCR transgenic mice express a monoclonal TCR specific for Salmonella
flagellin peptide (427–441)-I-Ab. These mice were backcrossed to RAG-2–
deficient C57BL/6 mice to obtain RAG-deficient SM1 offspring that contained SM1 CD4 T cells but no other lymphocytes. Mice used for experiments were 6–12 wk old, unless otherwise noted. All animal experiments
were approved by the University of California, Davis, Institutional Animal
Care and Use Committee.
using biotinylated anti–IFN-g (BD Biosciences), AKP Streptavidin (BD
Biosciences), and 1-Step NBT/BCIP substrate (Thermo Scientific, Waltham, MA). Cytokine spots were counted using an ImmunoSpot S5 Core
Analyzer (C.T.L., Shaker Heights, OH), and the total number of IFN-g–
producing CD4+ T cells per spleen was calculated.
Salmonella and Chlamydia infection
Mice were infected and treated with antibiotics as described in the previous
paragraph. After RBC lysis, 1 million splenocytes were incubated in the
presence of 10 mM Salmonella-specific peptide (SseI, SseJ, FliC, or PagC;
Table I) or serial diluted HK S. typhimurium in 96-well round-bottom
plates. After 24–48 h of incubation at 37˚C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) precoated
with purified anti–IFN-g, anti–IL-2, or anti–IL-4 (eBiosciences). Cytokine
production was detected using biotinylated anti–IFN-g, anti–IL-2, or anti–
IL-4 (eBiosciences), respectively, followed by ExtrAvidin-Peroxidase
substrate (Sigma-Aldrich). Plates were analyzed using a spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA), and cytokine
concentrations were calculated according to standard curves.
Flow cytometry
Spleens were harvested from naive and infected mice. Single-cell preparations were made using PBS with 2% FCS. Aliquots of single-cell suspension were stained with a panel of Abs (listed below) and analyzed on
a FACSCanto or an LSRFortessa flow cytometer (BD Biosciences, San Jose,
CA). Abs used included FITC-CD71, APC-Ter119, eFlour450-CD3, FITCCD11a, APC–IFN-g, PE-CD45.1, APC-eF780-CD45R, APC-CD19, FITCCD69, PE-CD62L, PE-Cy7-CD44, APC-Cy7-CD25 (eBioscience, San
Diego, CA), and PerCP-CD4 and APC-Cy7-CD8 (BD Biosciences, San
Diego, CA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Salmonella-specific serum Ab ELISA
Mice were bled retro-orbitally at various time points postinfection. Serum
was collected and analyzed by ELISA for Salmonella-specific Ab, as
previously described (13). Briefly, serial dilutions of serum samples were
added to heat-killed (HK) S. typhimurium-coated microtiter plates (Costar,
Corning, NY). Salmonella-specific Abs were detected using biotinylated
isotype-specific Abs (eBioscience) and ExtrAvidin-Peroxidase substrate
(Sigma-Aldrich, St. Louis, MO).
Salmonella-specific ELISPOT assay
At 2 wk after Salmonella infection, mice were treated with enrofloxacin
(Baytril) at 2 mg/ml in drinking water to eradicate bacteria. After removal
of antibiotic water, mice were rested for an additional week before spleens
were harvested and single-cell suspensions prepared. After RBC lysis,
CD4+ T cells were enriched via magnetic selecting LS MACS columns and
CD4 magnetic beads (Miltenyi Biotec, Auburn, CA). Purified CD4+ T cells
were coincubated with irradiated APCs of naive mice in the presence of
10 mM Salmonella-specific peptide (SseI, SseJ, FliC, or PagC; Table I) in
96-well ELISPOT plates (Millipore, Billerica, MA) precoated with purified
anti–IFN-g (BD Biosciences). SseI, SseJ, and FliC epitopes were previously described (38). The PagC MHC class II epitope was identified with
an overlapping peptide screen using a PagC-specific T cell line. After 20 h
of incubation at 37˚C, cells were washed and cytokine spots developed
Table I. Salmonella-specific MHC class II epitopes
Salmonella
Peptide
FliC 427–441
SseI 268–280
SseJ 329–341
PagC 163–174
Salmonella Protein
Epitope Sequence
Bacterial flagellum
SPI2-secreted
effector protein
SPI2-secreted
effector protein
phoP-activated gene
C
VQNRFNSAITNLGNT
LIYYTDFSNSSIA
CYYETADAFKVIM
GYEGSNISSTKI
Generation of bone marrow chimeras
Bone marrow cells from donor mice were isolated under sterile conditions,
and 1 3 106 donor cells were injected i.v. into recipient mice via the tail
vein. Recipient mice were given 1000 rads irradiation 16 h before injection, using a cesium irradiator. Recipient mice were treated with antibiotic
water for the first 6 wk after bone marrow reconstitution, and chimerism
was used at 8 wk after reconstitution.
DC enrichment and in vitro stimulation
Salmonella flagellin was purified as described previously (39). Flagellin
peptide (flagellin427–441) was purchased from Invitrogen (Carlsbad, CA).
Spleens were harvested and digested with collagenase D (Roche Diagnostics, Indianapolis, IN) (39), and DCs were enriched to .85–95% purity
via magnetic selecting LS MACS column and CD11c magnetic beads
(Miltenyi Biotec). Enriched DCs were seeded at 1 3 105 cells per well in
a 96-well plate and cocultured with 1 3 105 purified SM1 T cells plus Ag.
SM1 T cells were recovered 16 h later, stained with surface markers, and
analyzed by flow cytometry.
Detection of C. muridarum-specific CD4 T cells
The C. muridarum-specific epitope PmpG-1 has been previously described
(40). PE-labeled MHC class II tetramers (I-Ab) containing C. muridarum
polymorphic membrane protein G-1 (PmpG-1) residue 303–311 were
made in our laboratory (to be published elsewhere). Single-cell preparations from spleens were incubated for 1 h at room temperature with 10 nM
PmpG-1 tetramer in Fc block. Tetramer-specific cells were enriched via
magnetic selecting LS MACS column and anti-PE magnetic beads (Miltenyi Biotec) (41). The enriched cells were stained with surface markers
and analyzed by flow cytometry.
Results
Sirpa2/2 mice exhibit a marked increase in splenic erythroid
cells
We first examined the splenic cellular profile of naive Sirpa2/2
mice. Consistent with previous reports, Sirpa2/2 mice exhibited
mild splenomegaly, with a total cell number ∼2-fold that of agematched WT controls (Fig. 1A) (30). Further analysis demonstrated that the increased number of splenic cells in Sirpa2/2
mice is mostly, if not completely, due to higher percentages and
total cell numbers of RBCs (CD712Ter119+) and RBC progenitors (CD71+Ter119+) (Fig. 1B, 1C). Notably, although we observed a marked increase in the erythrocyte frequency and total
cell number in the spleen of Sirpa2/2 mice, we did not detect any
difference in the bone marrow, indicating that erythropoiesis in the
bone marrow of Sirpa2/2 mice is normal (Fig. 1B). The marked
increase in RBCs in Sirpa2/2 mice also correlated with decreased
lymphocyte cell numbers. CD4 T cell, CD8 T cell, and B cell
numbers were ∼2-fold reduced in Sirpa2/2 naive mice compared with WT controls (Fig. 1D, 1E). These results explain the
previous finding that splenic white pulps were reduced in Sirpa2/2
mice (30, 33).
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S. typhimurium strain BRD509 (aroA2aroD2) was grown overnight in
Luria-Bertani broth without shaking, and bacterial concentration was estimated using a spectrophotometer (OD, 600 nm). Chlamydia muridarum
strain Nigg (American Type Culture Collection, Manassas, VA) was cultivated, purified, aliquoted, titered, and stored at 280˚C, as previously
described (37). Mice were infected i.v. in the lateral tail vein with 5 3 105
CFU S. typhimurium or 1 3 105 inclusion forming units of C. muridarum
diluted in 200 ml PBS. The actual Salmonella bacterial dose administered
was always confirmed by plating serial dilutions of the bacterial culture
onto MacConkey agar plates. A fresh-thawed Chlamydia aliquot from the
same stock was used for each experiment. For survival studies, mice were
monitored daily for signs of illness and sacrificed at the moribund stage. To
determine Salmonella bacterial loads in vivo, spleens and livers were removed from infected mice at various time points. Serial dilutions of organ
homogenates were plated on MacConkey agar plates, and bacterial counts
per organ were calculated.
Salmonella-specific cytokine ELISA
The Journal of Immunology
2539
SIRPa is indispensable for protective immunity to
S. typhimurium infection
To examine the role that SIRPa plays in Salmonella resistance, we
infected WT, Sirpa+/2, and Sirpa2/2 mice (littermates) i.v. with
5 3 105 attenuated S. typhimurium (BRD509) and monitored
infected mice for signs of disease. Given the previous association
between EPO and susceptibility to Salmonella infection (5), we
hypothesized that increased erythropoiesis in Sirpa2/2 mice
would heighten susceptibility to Salmonella infection. Indeed,
although WT and Sirpa+/2 mice survived for months after Salmonella infection, all Sirpa2/2 mice died between 2 and 4 wk
postinfection (Fig. 2A). The inability of Sirpa2/2 mice to resolve
Salmonella infection was mapped to a deficiency in hematopoietic
cell lineages because Sirpa2/2 bone marrow chimeras exhibit
a similar survival pattern (Fig. 2B).
We next accessed the bacterial burden in WT and Sirpa2/2 mice
at various time points after Salmonella infection. WT C57BL/6
mice typically resolve attenuated Salmonella infection ∼5 wk
postinfection. At 1 wk postinfection, Sirpa2/2 mice have a
slightly higher bacterial burden than do WT mice in both spleen
and liver (Fig. 2C, 2D). This small, but statistically significant,
difference indicates that innate immune response in Sirpa2/2
mice is impaired in cleaning bacterial infection. A much greater
difference in bacterial counts between WT and Sirpa2/2 mice
became obvious as infection progressed to week 2 (Fig. 2C, 2D).
Although many Sirpa2/2 mice died between 2 and 3 wk, surviving Sirpa2/2 mice exhibited a dramatic increase in Salmonella
colonization in both spleen and liver at 3 wk postinfection (Fig.
2C, 2D). In conclusion, Sirpa2/2 mice fail to control Salmonella
replication in vivo and ultimately succumb to an infection that is
self-limiting in WT controls.
Defects in Salmonella-specific CD4 T cell response in Sirpa2/2
mice
Our laboratory and others have previously documented a similar
inability to resolve Salmonella infection in MHC class II-deficient,
CD282/2, and T-bet2/2 mice (8, 10, 13), suggesting that SIRPa
deficiency may affect the CD4 T cell response to Salmonella infection. To test this hypothesis, we examined Salmonella epitopespecific Th1 responses in Sirpa2/2 mice, using IFN-g ELISPOTs
and four different natural Salmonella epitopes (Table I). To allow
time for Salmonella-specific CD4 T cell responses to develop
in vivo while keeping Sirpa2/2 mice alive, mice were treated with
antibiotics during weeks 3 and 4 postinfection. At 5 wk after
initial infection, we observed that SseI-, SseJ-, FliC-, and PagCspecific CD4 T cell responses were diminished in Sirpa2/2 mice
(Fig. 3A). In contrast, WT mice maintained an elevated frequency
of CD4 T cells specific for each of these Salmonella epitopes (Fig.
3A).
It was possible that CD4 T cells in Sirpa2/2 mice produced
other Th1 cytokines such as IL-2 instead of IFN-g, or that they
polarized down the Th2 pathway by producing IL-4. To test these
possibilities, we used cytokine ELISA to measure IFN-g, IL-2,
and IL-4 production in response to Salmonella Ags. Similar to
what we discovered in IFN-g ELISPOT assays, Sirpa2/2 mice
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FIGURE 1. Increased splenic erythropoiesis in
Sirpa2/2 naive mice. (A) Total splenocytes recovered from the spleens of WT and Sirpa2/2
mice. (B) The percentages of RBCs (Ter119+
CD712) and RBC precursors (Ter119+CD71+) in
the bone marrow and spleen of WT and Sirpa2/2
mice, as measured by flow cytometry. (C) Total
RBCs and RBC precursors recovered from the
spleens of WT and Sirpa2/2 mice. (D) Total CD4
and CD8 T lymphocytes recovered from the spleen
of WT and Sirpa2/2 mice. (E) Total B lymphocytes
recovered from the spleen of WT and Sirpa2/2
mice. Data shown are representative of three similar experiments. Error bars represent the mean 6
SEM, **p , 0.01.
2540
ROLE OF SIRPa IN SALMONELLA INFECTION
no IL-4 production was detected, regardless of the dose of HK
S. typhimurium used (data not shown).
Taken together, we conclude that Sirpa2/2 mice develop an Agspecific deficiency in CD4 T cell responses to single Salmonella
Ags (SseI, SseJ, flagellin, and PagC), and the overall polyclonal
Salmonella-specific Th1 response is also reduced.
SIRPa deficiency on DCs leads to a defect in Ag presentation
Defects in Ab response in Sirpa2/2 mice
Although Salmonella is an intracellular bacterium, it is known that
Ab responses play an important role in protective immunity (11–
13). We therefore examined Salmonella-specific serum Ab postinfection responses of WT and Sirpa2/2 mice. Both WT and
Sirpa2/2 mice developed elevated titers of Salmonella-specific
IgM and IgG2c at 2 and 3 wk postinfection; however, the overall
Ab titers were lower in Sirpa2/2 mice than in WT controls (Fig.
5A–D). No Salmonella-specific IgG1 response was detected in
either WT or Sirpa2/2 mice (data not shown). These data show
that mice deficient in SIRPa generate Salmonella-specific Ab
responses after Salmonella infection but that these responses are
lower than those in WT mice.
FIGURE 2. SIRPa expression is required for survival after Salmonella
infection. (A) Survival curve of Sirpa2/2 , Sirpa+/2 , and WT littermate
control mice infected i.v. with 5 3 105 live attenuated Salmonella (BRD509).
Data shown are combined results from three independent experiments, with
at least 10 mice per group. (B) Survival curve of WT and Sirpa2/2 bone
marrow chimeras infected i.v. with 5 3 105 live attenuated Salmonella
(BRD509). Data represent at least five mice per group. (C and D) Spleens (C)
and livers (D) from infected WT and Sirpa2/2 mice were recovered 1, 2, and
3 wk postinfection. The bacterial counts per organ were determined by serial
dilution and plating. Data shown are representative of three independent
experiments. Error bars represent the mean 6 SEM. **p , 0.01.
produced less IFN-g and IL-2 overall (Fig. 3B, 3C). In contrast,
none of the mice produced IL-4, indicating that CD4 T cells do
not skew toward the Th2 lineage in either WT or Sirpa2/2 mice
(data not shown).
To assess the total polyclonal Salmonella-specific CD4 T cell
response to Salmonella Ags, we next measured cytokine production in response to HK S. typhimurium. Similar to single-Ag
stimulations, HK S. typhimurium induced high levels of IFN-g
and IL-2 production by WT mice, whereas these cytokines were
only marginally produced by Sirpa2/2 mice (Fig. 3D, 3E). Again,
SIRPa deficiency does not affect the development of
Chlamydia-specific CD4 T cells
We next asked whether the failure to develop pathogen-specific
CD4 T cell responses in Sirpa2/2 mice was a peculiar feature
of Salmonella infection. We infected WT and Sirpa2/2 mice i.v.
with 1 3 105 C. muridarum, another intracellular bacterium that
requires a Th1 response for resolution (42, 43). Of interest, when
comparing Chlamydia epitope-specific CD4 T cell responses in
WT and Sirpa2/2 mice, using MHC class II tetramers, we did not
observe any defect in Sirpa2/2 mice (Fig. 6A, 6B). Indeed,
Sirpa2/2 mice developed similar clonal expansion of Chlamydia
(PmpG-1)-specific CD4 T cells following infection. Therefore, the
defects in CD4 T cell responses we detected in Salmonella infection were not universal but appear restricted to the CD4 T cell
response to Salmonella. Together, these data point to a role for
SIRPa in the generation of Salmonella-specific Th1 responses and
the regulation of erythropoiesis (Fig. 7).
Discussion
In an earlier study, we reported a marked increase in splenic
erythropoiesis after systemic Salmonella infection of mice (5). We
concluded that massive expansion of erythrocytes accounts for
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SIRPa is expressed by a subpopulation of APCs in mice (22). It
seemed possible that the defect we detected in peptide-specific
CD4 T cell responses in Sirpa2/2 mice was due to the lack of
SIRPa expression by DCs. To test this hypothesis, we examined
the ability of SIPRa-deficient DCs to activate Ag-specific CD4
T cells in vitro. We cocultured Salmonella flagellin-specific SM1
T cells (35) with WT or SIPRa-deficient DCs in the presence or
absence of crude (flagellin) or processed (flagellin peptide) Ag. At
16 h after coincubation, SM1 T cells upregulated the activation
markers CD69, CD44, and CD25 and downregulated CD62L in
a dose-dependent manner when flagellin was processed and presented by WT DCs (Fig. 4A–D). However, the activation level of
SM1 T cells was considerably lower in the presence of SIRPadeficient DCs when compared with WT DCs (Fig. 4A–D), indicating that SIRPa-deficient DCs failed to process and present
flagellin to the same level as WT DCs. In contrast, when SM1
T cells were activated by flagellin peptide, expression of SIRPa on
DCs was not essential (Fig. 4A–D). With all these findings taken
together, we conclude that SIPRa expression on DCs plays
a crucial role in Ag processing and presentation to CD4 T cells.
The Journal of Immunology
2541
Salmonella-induced splenomegaly and also increased susceptibility to infection (5). As a key regulatory molecule for peripheral
RBC turnover, SIRPa is widely expressed on myeloid lineage
cells such as macrophages (27). Consistent with previous observations (30), we found that Sirpa2/2 mice exhibit an increase in
the number of erythroid cells in the spleen. Surprisingly, we found
that Sirpa2/2 mice are considerably more susceptible to Salmonella infection, as demonstrated by uncontrolled bacterial replication and increased mortality after challenge with attenuated
bacteria. Thus, our current study provides another example of
FIGURE 4. Reduced activation of Salmonellaspecific CD4 T cells by SIRPa-deficient DCs
in vitro. CD11c+ DCs (1 3 105) purified from WT
or SIRPa-deficient mice were cocultured with 1 3
105 flagellin-specific SM1 T cells in the presence
or absence of various doses of Ag flagellin or 10
mg/ml flagellin peptide. Graphs depict the percent
expression of CD69 (A), CD44 (B), CD25 (C), or
loss of CD62L (D) on gated SM1 T cells 16 h
poststimulation. Error bars represent mean 6 SEM.
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FIGURE 3. Impaired Ag-specific
CD4+ T cell response in Sirpa2/2
mice after Salmonella infection. WT
and Sirpa2/2 mice were infected i.v.
with 5 3 105 attenuated Salmonella
(BRD509) for a total of 5 wk. All mice
were treated with antibiotics during
weeks 3 and 4. (A) Purified splenic
CD4+ T cells from infected mice were
cocultured with Salmonella-specific
CD4 T cell Ag (SseJ, SseI, FliC, or
PagC) in the presence of irradiated
APCs for 20 h in an ELISPOT plate
precoated with anti–IFN-g. Ag-specific CD4 T cell numbers were determined. Data represent three similar
experiments with at least three mice
per group. (B and C) A total of 1 million splenocytes from infected WT and
Sirpa2/2 mice were cocultured with
Salmonella-specific CD4 T cell Ags
(SseJ, SseI, FliC or PagC) for 24–48 h.
Cytokine productions in culture supernatants were determined by cytokine ELISA. Data represent three
similar experiments with at least three
mice per group. (D and E) A total of 1
million total splenocytes from infected
WT and Sirpa2/2 mice were cocultured with serial diluted HK S. typhimurium for 24–48 h. Cytokine productions in culture supernatants were
determined by cytokine ELISA. Data
represent three similar experiments
with at least three mice per group.
2542
ROLE OF SIRPa IN SALMONELLA INFECTION
FIGURE 5. Salmonella-specific Ab responses in
WT and Sirpa2/2 mice. WT and Sirpa2/2 mice were
infected i.v. with 5 3 105 attenuated Salmonella
(BRD509). Serum was collected 2 (A, B) and 3 (C, D)
wk postinfection (p.i.). Salmonella-specific IgM (A, C)
and IgG2c (B, D) titers were determined by ELISA.
Graphs show OD readings (mean 6 SEM) of IgM
and IgG2c responses. Data represent three independent experiments with at least four mice per group.
FIGURE 6. SIRPa expression is not required for Ag-specific CD4 T cell
response to C. muridarum infection. (A) FACS plots of PmpG-1 MHC
class II tetramer-specific CD4 T cell staining in naive, WT, and Sirpa2/2
mice 5 wk after C. muridarum i.v. infection. (B) Total PmpG-1–specific
CD4 T cell numbers in WT and Sirpa2/2 mice.
known Salmonella epitopes: SseI, SseJ, FliC, and PagC. These
epitopes represent the only well-defined epitopes in the Salmonella mouse model and therefore suggested a broad reduction in
the Th1 response to Salmonella (38, 44). The overall Th1 cytokine
production by Sirpa2/2 mice is also largely diminished, as
stimulation of Sirpa2/2 splenocytes with single or bulk Salmonella Ag (HK S. typhimurium) leads to only minimal IFN-g or IL2 production. Indeed, the survival curve of Sirpa2/2 mice after
Salmonella infection parallels that observed in mice lacking the
Th1-specific transcription factor T-bet, suggesting a similar deficiency in these mouse models (10). Similar requirement for
SIRPa in induction of Th1 responses has also been reported in
a recent study using the parasite Leishmania major infection
model (45).
In addition to macrophages, SIRPa is also abundantly expressed
on DCs (22). CD8a2CD11c+ DCs in the spleen express a much
higher level of SIRPa than do CD8a+CD11c+ DCs (26, 46, 47).
Lack of expression of SIRPa or its ligand, CD47, results in reduced numbers of CD8a2CD11c+ DCs (26). Thus, it is possible
that the diminished Ag-specific CD4 T cell responses in Sirpa2/2
mice are due to the reduced number of this CD8a2CD11c+ DC
population and/or less efficient Ag presentation by SIRPa-deficient DCs. Indeed, our data clearly show that the ability of Agspecific CD4 T cell priming by SIRPa-deficient DCs in vitro was
reduced when compared with that of WT DCs. These results
provide a mechanistic explanation for the reduced Salmonella Agspecific CD4 T cell response in Sirpa2/2 mice in vivo. Similar
FIGURE 7. Predicted model of how SIRPa deficiency leads to increased
susceptibility to Salmonella infection.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a correlation between increased erythropoiesis and increased susceptibility to Salmonella infection.
There are likely to be multiple mechanisms by which erythropoiesis and SIRPa affect susceptibility to Salmonella (Fig. 7). We
previously demonstrated that increased EPO production encourages bacterial growth and speculated that this resulted from an
alteration in the architecture of the spleen that inhibited the
adaptive immune response (5). An alternative possibility is that
EPO can directly inhibit macrophage bactericidal activity, thus
increasing susceptibility to Salmonella (14). Our new data suggest
that SIRPa deficiency also induces erythropoiesis, impairs host
immunity, and encourages Salmonella growth. Together these
processes suggest a growing link between erythroid development
and susceptibility to bacterial infection.
The increased susceptibility of SIRPa-deficient mice to bacterial infection may also be independent of increased erythropoiesis
and directly caused by defects in Ag-specific CD4 T cell responses. Our ELISPOT results show that 5 wk post-Salmonella
infection, Sirpa2/2 mice lack Ag-specific CD4 T cells of all
The Journal of Immunology
in vitro studies have also reported that interaction of SIRPa on
DCs with CD47 on T cells contributes to the activation of Agspecific cytotoxic T cells (25).
In addition to reduced cell-mediated immunity, the development
of Salmonella-specific Ab responses was also reduced in Sirpa2/2
mice. As described earlier, increased erythropoiesis in Sirpa2/2
mice disrupts the splenic architecture after Salmonella infection.
The enlarged spleen could lead to disruption of efficient B cell and
T helper cell interactions, thus making Ab production less efficient.
Overall, our present study demonstrates an indispensable role for
SIRPa in protective immunity against Salmonella infection (Fig.
7). Deficiency of SIRPa correlated with a reduction of CD4 Th1
responses to Salmonella-specific Ags, inefficient Ag presentation,
and reduced Salmonella-specific Ab. Our data also strengthen the
association between susceptibility to Salmonella and erythroid
development and suggest that focusing on this interaction may
assist the development of vaccines or therapeutics for bacterial
infection.
We thank Dr. S.S. Way (University of Minnesota) for helpful discussion.
Disclosures
The authors have no financial conflicts of interest.
References
1. Tam, M. A., A. Rydström, M. Sundquist, and M. J. Wick. 2008. Early cellular
responses to Salmonella infection: dendritic cells, monocytes, and more.
Immunol. Rev. 225: 140–162.
2. Ravindran, R., and S. J. McSorley. 2005. Tracking the dynamics of T-cell activation in response to Salmonella infection. Immunology 114: 450–458.
3. Parry, C. M., T. T. Hien, G. Dougan, N. J. White, and J. J. Farrar. 2002. Typhoid
fever. N. Engl. J. Med. 347: 1770–1782.
4. Griffin, A. J., and S. J. McSorley. 2011. Development of protective immunity to
Salmonella, a mucosal pathogen with a systemic agenda. Mucosal Immunol. 4:
371–382.
5. Jackson, A., M. R. Nanton, H. O’Donnell, A. D. Akue, and S. J. McSorley. 2010.
Innate immune activation during Salmonella infection initiates extramedullary
erythropoiesis and splenomegaly. J. Immunol. 185: 6198–6204.
6. Srinivasan, A., J. Foley, and S. J. McSorley. 2004. Massive number of antigenspecific CD4 T cells during vaccination with live attenuated Salmonella causes
interclonal competition. J. Immunol. 172: 6884–6893.
7. Mittrücker, H. W., A. Köhler, and S. H. Kaufmann. 2002. Characterization of the
murine T-lymphocyte response to Salmonella enterica serovar Typhimurium
infection. Infect. Immun. 70: 199–203.
8. Hess, J., C. Ladel, D. Miko, and S. H. Kaufmann. 1996. Salmonella typhimurium
aroA- infection in gene-targeted immunodeficient mice: major role of CD4+
TCR-alpha beta cells and IFN-gamma in bacterial clearance independent of
intracellular location. J. Immunol. 156: 3321–3326.
9. Nauciel, C. 1990. Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection. J. Immunol. 145: 1265–
1269.
10. Ravindran, R., J. Foley, T. Stoklasek, L. H. Glimcher, and S. J. McSorley. 2005.
Expression of T-bet by CD4 T cells is essential for resistance to Salmonella
infection. J. Immunol. 175: 4603–4610.
11. Mastroeni, P., C. Simmons, R. Fowler, C. E. Hormaeche, and G. Dougan. 2000.
Igh-6(2/2) (B-cell-deficient) mice fail to mount solid acquired resistance to oral
challenge with virulent Salmonella enterica serovar typhimurium and show
impaired Th1 T-cell responses to Salmonella antigens. Infect. Immun. 68: 46–53.
12. Mittrücker, H. W., B. Raupach, A. Köhler, and S. H. Kaufmann. 2000. Cutting
edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection. J. Immunol. 164: 1648–1652.
13. McSorley, S. J., and M. K. Jenkins. 2000. Antibody is required for protection
against virulent but not attenuated Salmonella enterica serovar typhimurium.
Infect. Immun. 68: 3344–3348.
14. Nairz, M., A. Schroll, A. R. Moschen, T. Sonnweber, M. Theurl, I. Theurl,
N. Taub, C. Jamnig, D. Neurauter, L. A. Huber, et al. 2011. Erythropoietin
contrastingly affects bacterial infection and experimental colitis by inhibiting
nuclear factor-kB-inducible immune pathways. Immunity 34: 61–74.
15. Koury, M. J., S. T. Sawyer, and S. J. Brandt. 2002. New insights into erythropoiesis. Curr. Opin. Hematol. 9: 93–100.
16. Elliott, S., E. Pham, and I. C. Macdougall. 2008. Erythropoietins: a common
mechanism of action. Exp. Hematol. 36: 1573–1584.
17. van Beek, E. M., F. Cochrane, A. N. Barclay, and T. K. van den Berg. 2005.
Signal regulatory proteins in the immune system. J. Immunol. 175: 7781–7787.
18. Fujioka, Y., T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi,
M. Tsuda, T. Takada, and M. Kasuga. 1996. A novel membrane glycoprotein,
SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase
SHP-2 in response to mitogens and cell adhesion. Mol. Cell. Biol. 16: 6887–
6899.
19. Sano, S., H. Ohnishi, A. Omori, J. Hasegawa, and M. Kubota. 1997. BIT, an
immune antigen receptor-like molecule in the brain. FEBS Lett. 411: 327–334.
20. Comu, S., W. Weng, S. Olinsky, P. Ishwad, Z. Mi, J. Hempel, S. Watkins,
C. F. Lagenaur, and V. Narayanan. 1997. The murine P84 neural adhesion
molecule is SHPS-1, a member of the phosphatase-binding protein family.
J. Neurosci. 17: 8702–8710.
21. Saginario, C., H. Sterling, C. Beckers, R. Kobayashi, M. Solimena, E. Ullu, and
A. Vignery. 1998. MFR, a putative receptor mediating the fusion of macrophages. Mol. Cell. Biol. 18: 6213–6223.
22. Lahoud, M. H., A. I. Proietto, K. H. Gartlan, S. Kitsoulis, J. Curtis, J. Wettenhall,
M. Sofi, C. Daunt, M. O’keeffe, I. Caminschi, et al. 2006. Signal regulatory
protein molecules are differentially expressed by CD82 dendritic cells.
J. Immunol. 177: 372–382.
23. Barclay, A. N., and M. H. Brown. 2006. The SIRP family of receptors and
immune regulation. Nat. Rev. Immunol. 6: 457–464.
24. Matozaki, T., Y. Murata, H. Okazawa, and H. Ohnishi. 2009. Functions and
molecular mechanisms of the CD47-SIRPalpha signalling pathway. Trends Cell
Biol. 19: 72–80.
25. Seiffert, M., P. Brossart, C. Cant, M. Cella, M. Colonna, W. Brugger, L. Kanz,
A. Ullrich, and H. J. Bühring. 2001. Signal-regulatory protein alpha (SIRPalpha)
but not SIRPbeta is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34(+)CD38(2) hematopoietic cells.
Blood 97: 2741–2749.
26. Okajo, J., Y. Kaneko, Y. Murata, T. Tomizawa, C. Okuzawa, Y. Saito, Y. Kaneko,
T. Ishikawa-Sekigami, H. Okazawa, H. Ohnishi, et al. 2007. Regulation by Src
homology 2 domain-containing protein tyrosine phosphatase substrate-1 of
alpha-galactosylceramide-induced antimetastatic activity and Th1 and Th2
responses of NKT cells. J. Immunol. 178: 6164–6172.
27. Barclay, A. N. 2009. Signal regulatory protein alpha (SIRPalpha)/CD47 interaction and function. Curr. Opin. Immunol. 21: 47–52.
28. Brown, E. J., and W. A. Frazier. 2001. Integrin-associated protein (CD47) and its
ligands. Trends Cell Biol. 11: 130–135.
29. Oldenborg, P. A., A. Zheleznyak, Y. F. Fang, C. F. Lagenaur, H. D. Gresham, and
F. P. Lindberg. 2000. Role of CD47 as a marker of self on red blood cells.
Science 288: 2051–2054.
30. Ishikawa-Sekigami, T., Y. Kaneko, H. Okazawa, T. Tomizawa, J. Okajo, Y. Saito,
C. Okuzawa, M. Sugawara-Yokoo, U. Nishiyama, H. Ohnishi, et al. 2006. SHPS1 promotes the survival of circulating erythrocytes through inhibition of
phagocytosis by splenic macrophages. Blood 107: 341–348.
31. Yamao, T., T. Noguchi, O. Takeuchi, U. Nishiyama, H. Morita, T. Hagiwara,
H. Akahori, T. Kato, K. Inagaki, H. Okazawa, et al. 2002. Negative regulation of
platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1. J. Biol. Chem. 277: 39833–39839.
32. Legrand, N., N. D. Huntington, M. Nagasawa, A. Q. Bakker, R. Schotte,
H. Strick-Marchand, S. J. de Geus, S. M. Pouw, M. Böhne, A. Voordouw, et al.
2011. Functional CD47/signal regulatory protein alpha (SIRP(alpha)) interaction
is required for optimal human T- and natural killer- (NK) cell homeostasis
in vivo. Proc. Natl. Acad. Sci. USA 108: 13224–13229.
33. Sato-Hashimoto, M., Y. Saito, H. Ohnishi, H. Iwamura, Y. Kanazawa, T. Kaneko,
S. Kusakari, T. Kotani, M. Mori, Y. Murata, et al. 2011. Signal regulatory protein
a regulates the homeostasis of T lymphocytes in the spleen. J. Immunol. 187:
291–297.
34. Saito, Y., H. Iwamura, T. Kaneko, H. Ohnishi, Y. Murata, H. Okazawa,
Y. Kanazawa, M. Sato-Hashimoto, H. Kobayashi, P. A. Oldenborg, et al. 2010.
Regulation by SIRPa of dendritic cell homeostasis in lymphoid tissues. Blood
116: 3517–3525.
35. McSorley, S. J., S. Asch, M. Costalonga, R. L. Reinhardt, and M. K. Jenkins.
2002. Tracking salmonella-specific CD4 T cells in vivo reveals a local mucosal
response to a disseminated infection. Immunity 16: 365–377.
36. Srinivasan, A., J. Foley, R. Ravindran, and S. J. McSorley. 2004. Low-dose
Salmonella infection evades activation of flagellin-specific CD4 T cells.
J. Immunol. 173: 4091–4099.
37. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purification and partial
characterization of the major outer membrane protein of Chlamydia trachomatis.
Infect. Immun. 31: 1161–1176.
38. Lee, S. J., J. B. McLachlan, J. R. Kurtz, D. Fan, S. E. Winter, A. J. Baumler,
M. K. Jenkins, and S. J. McSorley. 2012. Temporal expression of bacterial
proteins instructs host CD4 T cell expansion and Th17 development. PLoS
Pathog. 8: e1002499.
39. Salazar-Gonzalez, R. M., A. Srinivasan, A. Griffin, G. Muralimohan, J. M. Ertelt,
R. Ravindran, A. T. Vella, and S. J. McSorley. 2007. Salmonella flagellin induces
bystander activation of splenic dendritic cells and hinders bacterial replication
in vivo. J. Immunol. 179: 6169–6175.
40. Yu, H., X. Jiang, C. Shen, K. P. Karunakaran, and R. C. Brunham. 2009. Novel
Chlamydia muridarum T cell antigens induce protective immunity against lung
and genital tract infection in murine models. J. Immunol. 182: 1602–1608.
41. Moon, J. J., H. H. Chu, J. Hataye, A. J. Pagán, M. Pepper, J. B. McLachlan,
T. Zell, and M. K. Jenkins. 2009. Tracking epitope-specific T cells. Nat. Protoc.
4: 565–581.
42. Farris, C. M., and R. P. Morrison. 2011. Vaccination against Chlamydia genital
infection utilizing the murine C. muridarum model. Infect. Immun. 79: 986–
996.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Acknowledgments
2543
2544
43. Roan, N. R., T. M. Gierahn, D. E. Higgins, and M. N. Starnbach. 2006. Monitoring the T cell response to genital tract infection. Proc. Natl. Acad. Sci. USA
103: 12069–12074.
44. McSorley, S. J., B. T. Cookson, and M. K. Jenkins. 2000. Characterization of
CD4+ T cell responses during natural infection with Salmonella typhimurium. J.
Immunol. 164: 986–993.
45. Morimoto, N., Y. Murata, S. Motegi, K. Suzue, Y. Saito, H. Okazawa,
H. Ohnishi, T. Kotani, S. Kusakari, O. Ishikawa, and T. Matozaki. 2010. Requirement of SIRPa for protective immunity against Leishmania major. Biochem. Biophys. Res. Commun. 401: 385–389.
ROLE OF SIRPa IN SALMONELLA INFECTION
46. Hagnerud, S., P. P. Manna, M. Cella, A. Stenberg, W. A. Frazier,
M. Colonna, and P. A. Oldenborg. 2006. Deficit of CD47 results in a defect
of marginal zone dendritic cells, blunted immune response to particulate
antigen and impairment of skin dendritic cell migration. J. Immunol. 176:
5772–5778.
47. Naik, S. H., A. I. Proietto, N. S. Wilson, A. Dakic, P. Schnorrer,
M. Fuchsberger, M. H. Lahoud, M. O’Keeffe, Q. X. Shao, W. F. Chen, et al.
2005. Cutting edge: generation of splenic CD8+ and CD82 dendritic cell
equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures. J.
Immunol. 174: 6592–6597.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017