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Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein α -Deficient Mice This information is current as 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 Subscription Permissions Email Alerts This article cites 47 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/189/5/2537.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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). Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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. 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