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Macrophage recognition of Salmonella enterica serovar Typhimurium leads to a cascade of signaling events, including the activation of Src family and Syk kinases and the production of reactive oxygen species (ROS), which are critical for host innate defense during early stages of bacterial infection. ROS production depends on the NADPH oxidase, but little is known about the innate immune receptors and proximal adapters that regulate Salmonella-induced ROS. Herein, we demonstrate that serovar Typhimurium induces ROS through a pathway that requires both triggering receptor expressed on myeloid cells 2 (TREM2) and DAP12. This pathway is highly analogous to the pathways utilized by Fc receptors and integrins to regulate ROS production. Oral infection of mice with serovar Typhimurium demonstrates that the DAP12-dependent pathway regulates cecal colonization during early stages of Salmonella infection. Thus, DAP12 is an important regulator of Salmonella-induced ROS production in macrophages, and TREM2 is essential for linking DAP12 to the innate response to serovar Typhimurium.
Regulation of reactive oxygen species (ROS) production is an important innate immune defense against microbial pathogens. In the early stages of Salmonella enterica serovar Typhimurium infection, ROS generation by NADPH oxidase is crucial for control of bacterial growth, and in vitro studies show that ROS constitute an important bactericidal effector mechanism for macrophages infected with serovar Typhimurium (38). Host defense in later stages of Salmonella infection involves reactive nitrogen species, inflammatory cytokines (especially tumor necrosis factor alpha and gamma interferon), and an adaptive T-cell response (18).
ROS production is mediated by the recruitment of cytosolic proteins to the phagosomal membrane to generate a functional NADPH oxidase (1). Several receptors, including Fc receptors (FcRs) and integrins, can trigger ROS production in response to microbial pathogens (3, 23). For each of these receptors, an immunoreceptor tyrosine-based activation motif (ITAM) is critical for initiation of downstream events. FcRs contain an ITAM in their cytoplasmic tail or require the ITAM-bearing adapter Fc gamma chain (Fcγ) for surface expression, phagocytosis, and ROS production (30, 36). Integrins trigger Syk phosphorylation and neutrophil ROS production through the ITAM-containing adapters DAP12 and Fcγ (22). Activation of an ITAM-associated receptor leads to phosphorylation by Src family kinases (SFK) of the tyrosine residues within the ITAM consensus sequence. Syk family kinases are consequently recruited and activated, inducing signaling through multiple downstream pathways, including phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC) activation (30, 35). In neutrophils, ROS generation has also been demonstrated to involve the adapter protein Vav, PI 3-kinase, Rho family GTPases, phospholipase C, PKC, and the actin cytoskeleton (5, 9, 10, 29).
Less is known about innate immune receptors that regulate ROS production. These include dectin-1, which recognizes 1,3-β-glucans in the cell wall of yeasts (4). Dectin-1 contains a noncanonical ITAM motif in its cytoplasmic tail, and cross-linking of dectin-1 leads to Syk phosphorylation independent of DAP12 and Fcγ (24, 36). This results in activation of the NADPH oxidase and ROS production (36). In neutrophils, carcinoembryonic antigen-related cellular adhesion molecule 3 has been identified as an ITAM-containing innate immune receptor for Neisseria gonorrhoeae that regulates Neisseria-induced ROS via a Syk-dependent mechanism (28).
Toll-like receptors (TLRs) have also been implicated in ROS production, and signaling through the TLR adapter MyD88 is required for NADPH oxidase assembly in response to gram-negative bacteria (16, 25, 26). The roles of specific TLRs in NADPH oxidase assembly and ROS generation in response to serovar Typhimurium are cell type dependent. For example, TLR4 is required for NADPH oxidase activation in human neutrophils infected with serovar Typhimurium (37), but loss of TLR4 does not affect the mouse peritoneal macrophage ROS response to serovar Typhimurium (16). Similarly, we find that the TLR4 ligand lipopolysaccharide (LPS) does not induce detectable levels of ROS in murine bone marrow-derived macrophages (data not shown). The amount of ROS generated through activation of TLR pathways is considerably less than levels that are commonly associated with antimicrobial activities and may be more relevant to potential signal transduction pathways (20).
Many pathogenic bacteria can trigger ROS production by macrophages in the absence of complement or antibodies, but the macrophage receptors and signaling pathways that regulate this process are unknown. Potential receptors include the integrin family of proteins. The β2 integrin chain CD18 has been shown to bind LPS and β-glucans (17, 39), making it a potential receptor. Triggering receptor expressed on myeloid cells 2 (TREM2) has also been shown to bind several bacteria and fungi (8), and cross-linking of TREM2 on macrophage cell lines results in NO release (7). Specific innate immune receptors and proximal signaling pathways that are required for the generation of ROS in response to serovar Typhimurium have not been described. In this study, we characterize the regulation of Salmonella-induced ROS production by murine macrophages, demonstrating a requirement for both TREM2 and its adapter protein DAP12.
Chemical inhibitors for SFK (pyrrolopyrimidine 2 [PP2]), Syk kinase (piceatannol and Syk inhibitor), PI 3-kinase (wortmannin), PKC (bis-indoyl-maleimide [BIM]), p38 mitogen-activated protein kinase (MAPK) (SB21290), MEK1 (PD8059), Jun N-terminal protein kinase (JNK) (JNK inhibitor 1), actin polymerization (cytochalasin D), and the proteasome (MG-132 and lactacystin) were purchased from Calbiochem and dissolved in dimethyl sulfoxide (DMSO). All other reagents were purchased from Invitrogen or Sigma, unless otherwise noted.
Mice were maintained in the animal facilities of the San Francisco Veterans Affairs Medical Center and the University of Washington. All experiments were performed in accordance with AAALC guidelines and were approved by the Veterans Affairs Animal Care Committee and the University of Washington IACUC. C57BL/6 mice were purchased from Simonsen (Gilroy, CA). Homozygous DAP12-deficient mice on a C57BL/6 background were previously described (14). Femurs from the Fcγ-deficient mice on a C57BL/6 background (31) and the DAP12/Fcγ double knockouts on a C57BL/6 background (22) were provided by Charles Alpers (University of Washington) and Lewis Lanier (University of San Francisco), respectively.
The bacterial strain BC840 (a green fluorescent protein [GFP]-expressing derivative of Salmonella enterica serovar Typhimurium SL1344) was a gift from Brad Cookson (University of Washington, Seattle, WA). The RAW 264.7 murine macrophage cell line was from ATCC. TREM2 high- and low-level-expressing derivatives of RAW 264.7 and RAW 264.7 lines expressing short hairpin RNA interference (RNAi) for TREM2 or the control construct have been described previously (14).
Bone marrow-derived macrophages were grown on petri dishes in RPMI 1640 with 10% fetal bovine serum (FBS; HyClone), penicillin, streptomycin, 5 mM l-glutamine, and 50 ng/ml human macrophage colony-stimulating factor (Peprotech), and the medium was refreshed on day 4. On day 7, the adherent bone marrow macrophages were lifted from the plates with phosphate-buffered saline (PBS) containing 2 mM EDTA. The cells were resuspended in antibiotic-free RPMI 1640 with 10% FBS, 5 mM l-glutamine, 50 ng/ml human macrophage colony-stimulating factor, and 50 ng/ml murine gamma interferon, plated at 105 cells per well in 100-μl volumes in white plastic 96-well tissue culture plates (Corning), and incubated overnight. The next day, the medium was removed, 50 μl prewarmed, antibiotic-free RPMI 1640 with 10% FBS and 100 μM luminol was added to each well, and cells were incubated for 30 to 60 min at 37°C. For inhibitor studies, the inhibitors were added to the wells at the indicated concentrations 15 min prior to addition of stimuli. DMSO alone was added to control wells. Indicated stimuli were added to wells in 50 μl of antibiotic-free medium, and the luminescence was recorded over a 120-min interval with a plate reader (Veritas).
Age- and gender-matched 10- to 12-week-old C57BL/6 and DAP12-deficient mice were orally infected with serovar Typhimurium, following published techniques (2). Briefly, mice were pretreated with streptomycin starting 3 days prior to infection to sterilize the intestinal tract. A calculated inoculum of 103 log-phase Salmonella enterica serovar Typhimurium BC840 cells in 100 μl was administered orogastrically via a gavage needle. The inoculum was confirmed by plating on selective MacConkey agar overnight at 37°C. To determine the count of viable Salmonella bacteria in the tissues, the mice were euthanized on day 3 of infection. The mesenteric lymph node chain and portions of the spleen, liver, and cecum were weighed and homogenized in 0.5 ml PBS-0.025% Triton X-100 (Polytron homogenizer). Serial dilutions of homogenates were made and plated on MacConkey agar overnight at 37°C to determine numbers of viable organisms. Numbers of CFU were then calculated per gram of tissue.
TREM2 fusion protein binding to stationary Salmonella strain BC840 or E. coli labeled with Alexa Fluor Bodipy (Molecular Probes) was determined by incubating 106 bacteria with 1 μg of recombinant TREM2B/Fc or TREM1/Fc (R&D Systems) at 4°C for 1 h in staining buffer (PBS-1% FBS). Bacteria were washed with staining buffer and then incubated with allophycocyanin-conjugated goat anti-human immunoglobulin G (IgG) F(ab′)2 (Jackson ImmunoResearch) at 4°C for 30 min. After two washes in staining buffer, binding of recombinant TREM proteins to Bodipy- or GFP-conjugated bacteria was detected by using a FACSCalibur flow cytometer (BD Biosciences).
Salmonella strain BC840 was cultured overnight at 37°C in LB with 50 μg/ml streptomycin. Bodipy-conjugated E. coli was purchased from Molecular Probes. The BWZ.36 (BWZ) mouse T-cell lymphoma line was kindly provided by N. Shastri (University of California, Berkeley, CA). BWZ cells expressing TREM2 were previously described and shown to bind E. coli in a TREM2-dependent fashion (8). BWZ or BWZ/TREM2A cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FBS, 25 μM 2-mercaptoethanol, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were washed with staining buffer, and 106 cells were incubated with bacteria at a ratio of 300 bacteria per cell at 4°C for 1 h. Cells were washed twice with staining buffer, and the number of bound GFP Salmonella or Bodipy E. coli bacteria was determined by flow cytometry.
The BWZ line contains a lacZ reporter construct under the control of four copies of the NFAT promoter element (27). A stable TREM2- and DAP12-dependent lacZ reporter line (TD4) was derived from BWZ by retroviral transduction of DAP12 and TREM2B constructs. Viral supernatant from cells transfected with the previously described pMXpie retroviral vectors containing full-length, FLAG-tagged TREM2B and DAP12 was used to transduce BWZ cells according to published methods (14). Cells were selected in 2 μg/ml puromycin (Sigma), and stable, puromycin-resistant subclones were screened by flow cytometry for the expression of TREM2 and DAP12 by using specific monoclonal antibodies. TD4 cells were seeded in triplicate into 96-well plates at 5 × 104 cells/well in RPMI supplemented with 1% FBS and 10 ng/ml phorbol myristate acetate (PMA). For TREM2 cross-linking, 106 TD4 cells were incubated with 1 μg of the previously described anti-TREM2 monoclonal antibody 78 for 15 min at room temperature prior to seeding (14). Goat anti-mouse IgG Fab-specific F(ab′)2 (Sigma) at 10 μg/106 cells was added to the anti-TREM2-coated TD4 wells. Cells were incubated at 37°C for 15 min with ionomycin (250 nM), dextran sulfate (0.05 μg/ml), or Salmonella strain BC840 at the bacterium/cell ratio indicated, followed by the addition of gentamicin for a final concentration of 50 μg/ml. Plates were incubated for an additional 16 h at 37°C in a 5% humidified CO2 atmosphere and washed once with PBS. lacZ activity was then determined by incubating the cells with 150 μM chlorophenol red-β-d-galactopyranoside in PBS supplemented with 100 mM 2-mercaptoethanol, 9 mM MgCl2, and 0.125% Nonidet P-40. After 4 h, absorbance was measured at 595 nm, with correction for background absorbance at 650 nm. Values were normalized by subtracting the absorbance values of wells treated with PMA alone.
Each experiment was performed at least three times, and representative results are shown. Values shown in Fig. Fig.3A3A are medians and quartiles, and statistical significance was determined by the Mann-Whitney test. Values in other figures are means and standard errors of the means.
We first used a panel of specific inhibitors to define the signaling pathways controlling Salmonella-induced ROS production in bone marrow-derived macrophages, comparing the effects of the inhibitors to effects on the well-characterized zymosan-induced ROS response. Inhibitors of Syk kinase (piceatannol), PI 3-kinase (wortmannin), PKC (BIM), SFK (PP2), and actin polymerization (cytochalasin) substantially inhibited ROS production in response to either serovar Typhimurium or zymosan (Fig. (Fig.11 and and2B).2B). Inhibition of MAPK pathways demonstrated only modest inhibition of ROS production (Fig. (Fig.2A),2A), with the most potent effect seen with the MEK1 inhibitor PD98059, which reduced the peak response by approximately 50%. Proteasome inhibitors had minimal effect (Fig. (Fig.1B),1B), suggesting that proteasome-dependent functions, such as the activation of NF-κB, contribute little to ROS production. There was no measurable loss of cell viability following treatment with the highest concentrations of PP2, BIM, Syk inhibitor, PD98059, JNK inhibitor 1, and cytochalasin D, as determined by trypan blue exclusion after 2 hours of treatment. Modest decreases in cell viability were seen for treatment with the highest concentrations of piceatannol (98%), SB21290 (95%), MG-132 (94%), lactacystin (94%), wortmannin (90%), and DMSO (86%). Overall, the inhibitors had very similar effects on Salmonella- and zymosan-induced ROS, suggesting that serovar Typhimurium induces the ROS response through a signaling pathway similar to that activated by zymosan. Zymosan activates ROS through dectin-1, which contains an atypical but functional ITAM (36). ITAM signaling activates Syk kinase, PI 3-kinase, SFK, PKC, and actin polymerization, consistent with our findings with inhibitors for these pathways (21, 33). The importance of these signaling intermediates suggests that serovar Typhimurium, like zymosan, induces ROS generation through an ITAM-mediated pathway in macrophages.
Known ITAM-containing molecules in macrophages include the conventional ITAM adapters Fcγ and DAP12 as well as dectin-1, which contains an unconventional ITAM motif that can nonetheless interact with Syk (24, 36). Inhibition of dectin-1 by laminarin (Fig. (Fig.3A)3A) had no effect on Salmonella-induced ROS, suggesting that dectin-1 does not regulate the macrophage ROS response to serovar Typhimurium. As anticipated, laminarin treatment substantially inhibited zymosan-induced ROS (Fig. (Fig.3A).3A). To determine whether conventional ITAM-bearing adapters contributed to Salmonella-induced ROS production, we tested bone marrow-derived macrophages from wild-type (WT), Fcγ−/−, DAP12−/−, and Fcγ/DAP12 double-deficient mice. Only bone marrow macrophages derived from mice deficient in DAP12 demonstrated substantially reduced (>90%) ROS production after stimulation with serovar Typhimurium (Fig. (Fig.3B).3B). In contrast, ROS response to PMA treatment did not differ significantly (Fig. (Fig.3C).3C). Thus, DAP12 is a critical ITAM adapter for the oxidative burst in macrophages exposed to serovar Typhimurium.
Based on the above-mentioned findings, we predicted that DAP12 contributes to the innate immune response to serovar Typhimurium. To test this hypothesis, we orally infected streptomycin-treated WT and DAP12-deficient mice with serovar Typhimurium. On day 3 postinfection, bacterial burden was significantly higher in the ceca of DAP12-deficient mice than in those of WT mice (Fig. (Fig.4A).4A). This was confirmed with immunofluorescence studies that demonstrated increased bacteria colonizing the ceca of DAP12-deficient mice relative to those in WT mice (Fig. (Fig.4B).4B). Histological examination at this time point also demonstrated increased inflammation in the ceca of DAP12-deficent mice (Fig. (Fig.4C).4C). A trend toward increased colonization of the spleen, liver, and mesenteric lymph nodes in the DAP12-defcient mice was noted, but these results did not meet statistical significance (Fig. (Fig.4A).4A). The differences between WT and DAP12-deficient mice were most pronounced in these early events, and no difference in overall survival was noted (data not shown).
DAP12 is an adapter protein that links several receptors to Syk in a variety of innate immune cells. In myeloid cells, DAP12 is known to associate with TREM1, TREM2, TREM3, signal-regulatory protein β1, and myeloid DAP12-associating lectin 1 (15, 33). In earlier work, we demonstrated that several types of bacteria can bind and activate the TREM2 receptor (8). Thus, TREM2 was an attractive candidate for the DAP12-associated receptor in the Salmonella-induced ROS pathway. To determine whether or not TREM2 contributes to Salmonella-induced ROS, we tested natural variants of the murine macrophage RAW 264.7 cell line that were isolated for low and high expression of TREM2 (14). Cells expressing low levels of TREM2 demonstrated decreased Salmonella-induced ROS compared to the line expressing high levels of TREM2, while response to zymosan was unaffected (Fig. 5A and B). Similar results were obtained using RNAi to knock down TREM2 expression in RAW 264.7 cells; TREM2 RNAi substantially reduced Salmonella-induced ROS without affecting the response to zymosan (Fig. (Fig.5C).5C). In contrast, the control RNAi demonstrated intact ROS responses to both serovar Typhimurium and zymosan (Fig. (Fig.5D).5D). Thus, the DAP12-associated receptor TREM2 contributes to Salmonella-induced ROS.
TREM2 has been shown to bind to E. coli as well as to several other species of bacteria through an interaction with polyanionic ligands (8). We therefore hypothesized that TREM2 may similarly act as a receptor for polyanionic ligands on Salmonella bacteria. However, in contrast to what was found for E. coli, soluble TREM2-Fc fusion protein failed to bind serovar Typhimurium (Fig. (Fig.6A).6A). Further, unlike E. coli, serovar Typhimurium did not bind to BWZ cells expressing high levels of surface TREM2 (Fig. (Fig.6B),6B), and serovar Typhimurium was unable to activate a TREM2/DAP12-dependent LacZ reporter system in BWZ cells (Fig. (Fig.6C).6C). Thus, although TREM2 is required for the generation of ROS by macrophages in response to serovar Typhimurium, this requirement does not appear to involve direct recognition of serovar Typhimurium by TREM2.
Our studies demonstrate that efficient Salmonella-induced ROS production by macrophages requires both DAP12 and TREM2. ROS are critical for defense against Salmonella infection, especially at early time points postinfection (18), and hosts lacking NADPH oxidase have increased bacterial colonization and more-rapid death after Salmonella infection (19). During oral infection of naïve mice, mucosal surfaces may rely in particular on DAP12-dependent innate responses to Salmonella. Our in vivo studies suggest that the DAP12-dependent pathway retards Salmonella growth at mucosal surfaces. We did not, however, detect significant differences in overall survival between DAP12-deficient mice and control mice. It is possible that there is redundancy in signaling adapter requirements for the ROS response to serovar Typhimurium in other cell types, such as neutrophils, causing the phenotype of the DAP12-deficient mice to be much milder than that of mice globally deficient in NADPH oxidase activity. In neutrophils, for example, Fcγ and DAP12 are both capable of mediating integrin-induced ROS (22).
It is also possible that the loss of DAP12 may enhance other defense mechanisms that compensate for the loss of ROS. DAP12 has been shown to inhibit TLR and FcR responses in macrophages and dendritic cells (6, 11), and it is possible that DAP12 may have similar effects on TLR or other responses in vivo. In a colitis model of Salmonella infection, mucosal inflammation has been shown to require MyD88-dependent signaling in hematopoietic cells (13). Thus, increased TLR responses in DAP12-deficient mice could contribute to the increase in mucosal inflammation seen in response to serovar Typhimurium and may partially offset defective ROS production by macrophages. The effect of DAP12 deficiency on the host response to other infections is complex, with some studies showing DAP12 deficiency to be protective, as in LPS-induced endotoxemia and Listeria monocytogenes infection (12, 34), and others showing increased host susceptibility to d-galactosamine-potentiated endotoxemia in the absence of DAP12 (12). Additionally, DAP12-independent pathways may effectively control systemic infection such that there is no difference in survival of DAP12-deficient mice. During the systemic phase of Salmonella infection, multiple host defense mechanisms are utilized, including complement activation, cytokine and chemokine production, and production of antigen-specific T cells and antibodies (18). The later phases may be able to compensate for defective ROS responses in macrophages, even if they are unable to compensate for a global loss of ROS response.
The ROS response to a variety of stimuli appears to depend on ITAM signaling pathways leading to Syk activation (22, 28, 36). With the exceptions of dectin-1 and carcinoembryonic antigen-related cellular adhesion molecule 3, the upstream innate immune receptors that trigger an ROS response to specific pathogens are unknown. TREM2 does not appear to be a direct receptor for serovar Typhimurium, although we cannot rule out the possibility that TREM2 can recognize serovar Typhimurium in the context of a coreceptor or in the presence of a specific host protein, similar to the requirement for LPS binding protein for TLR4 recognition of LPS. We hypothesize that TREM2 plays an intermediary role, connecting a yet-to-be-identified receptor for serovar Typhimurium to DAP12 and its signaling cascade (Fig. (Fig.7).7). Such a role for TREM2 has been demonstrated for the response of plexin-A1 to its ligand Sema6D on dendritic cells. TREM2 associates with plexin-A1, and loss of either TREM2 or DAP12 reduces the dendritic cell response to Sema6D (32). Similarly, CD18 does not associate directly with either DAP12 or Fcγ, yet they are required for integrin-mediated ROS in neutrophils, suggesting that a DAP12/Fcγ-associated receptor may act as an intermediary (22). Alternatively, TREM2 may interact with ligands on the macrophages themselves in a manner that promotes the generation of ROS but that we could not detect in our assays. In all, Syk activation via ITAM-containing or DAP12/Fcγ-associated receptors may be a common and essential pathway for the ROS response to a variety of pathogens.
In conclusion, our studies demonstrate that TREM2 and DAP12 are important in the innate immune response to serovar Typhimurium; they are critical for efficient ROS production by macrophages and for control of cecal colonization during oral infection in mice.
This work was supported by grants from the National Institutes of Health (R01 CA087922 and R01 AI062859), an Arthritis Foundation Northern California Chapter postdoctoral fellowship (J.F.C.), and the Veterans Administration (M.B.H.).
We thank Erene Niemi for technical assistance, Mike Daws for discussion of bacterial binding assays, and Charles Alpers and Lewis Lanier for providing mouse strains.
Editor: B. A. McCormick
Published ahead of print on 7 April 2008.