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Originally published online as doi:10.1189/jlb.0407255 on August 14, 2007

Published online before print August 14, 2007
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(Journal of Leukocyte Biology. 2007;82:1062-1069.)
© 2007 by Society for Leukocyte Biology

Securinine, a GABAA receptor antagonist, enhances macrophage clearance of phase II C. burnetii: comparison with TLR agonists

Kirk Lubick, Miranda Radke and Mark Jutila1

Veterinary Molecular Biology, Montana State University, Bozeman, Montana, USA

1 Correspondence: Veterinary Molecular Biology, Montana State University, 960 Technology Blvd., Bozeman, MT 59718, USA. E-mail: uvsmj{at}montana.edu

ABSTRACT

Innate immune cell stimulation represents a complementary approach to vaccines and antimicrobial drugs to counter infectious disease. We have used assays of macrophage activation and in vitro and in vivo phase II Coxiella burnetii infection models to compare and contrast the activity of a novel innate immune cell agonist, securinine, with known TLR agonists. As expected, TLR agonists, such as LPS (TLR4) and fibroblast-stimulating lipopeptide-1 (FSL-1; TLR2), induced macrophage activation and increased macrophage killing of phase II C. burnetii in vitro. FSL-1 also induced accelerated killing of C. burnetii in vivo. Securinine, a {gamma}-aminobutyric acid type A receptor antagonist, was found to induce TLR-independent macrophage activation in vitro, leading to IL-8 secretion, L-selectin down-regulation, and CD11b and MHC Class II antigen up-regulation. As seen with the TLR agonists, securinine also induced accelerated macrophage killing of C. burnetii in vitro and in vivo. In summary, as predicted by the literature, TLR agonists enhance macrophage killing of phase II C. burnetii in vitro, and at least for TLR2 agonists, this activity occurs in vivo as well. Securinine represents a novel macrophage agonist, which has similar effects as TLR agonists in this model yet apparently, does not act through known TLRs. Securinine has minimal toxicity in vivo, suggesting it or structurally similar compounds may represent novel, therapeutic adjuvants, which increase resistance to intracellular pathogens.

Key Words: adjuvant therapy • innate immunity • antimicrobial drugs • infectious disease • leukocyte

INTRODUCTION

Stimulation of the innate immune system leads to increased resistance to viral and bacterial infection [1 2 3 ]. TLR agonists are potent activators of innate immune cells, and when given in vivo, some induce increased resistance to infections, such as influenza and Listeria monocytogenes [3 4 5 ]. These results suggest that use of TLR agonists or other compounds, which stimulate innate immune cells, may provide effective therapeutic and/or prophylactic approaches to counter infectious diseases, for which there is currently a lack of vaccines and/or antimicrobial agents [6 ]. Furthermore, use of such adjuvants in combination with vaccines may provide protection against the vaccine-targeted pathogen prior to development of an effective adaptive immune response. This would be of considerable value for vaccines, which require multiple boosts such as the current anthrax vaccine [7 ]. Finally, selective enhancement of innate immunity might be of considerable benefit to patients whose adaptive immune system is compromised, such as AIDS patients and patients undergoing anti-cancer therapy. As such, there is an interest in developing compounds, which increase innate immune responses, as "drugs" that can be used therapeutically or prophylactically to enhance innate resistance to infection.

Our long-term goal is to identify novel, safe adjuvant drug formulations, which have activity in reducing intracellular, bacterial infections. We have used induced cytokine production and adhesion molecule expression as markers of macrophage activation to screen for novel agonists, which may activate cells in a TLR-independent manner and increase their killing of intracellular bacteria. Our assay for intracellular bacterial killing uses a biosafety level 2 (BSL-2; avirulent phase II) variant of the obligate intracellular pathogen Coxiella burnetii, the causative agent of Q-fever [8 ]. Phase II C. burnetii readily infects macrophages in vitro and in vivo, and accelerated killing of the bacterium in vitro is induced by TLR2 and TLR4 agonists [9 , 10 ]. Although normal mice eventually clear phase II C. burnetii infection, this is in part a result of an eventual adaptive immune response against the bacterium [11 ]. After injection of 1 x 107–1 x 108 phase II C. burnetii i.p. into normal mice, the bacterium spreads to the spleen and liver. An effective adjuvant in this model would act early (within the first few days after infection) and reduce the spread and enhance the killing of the bacterium prior to engagement of the adaptive immune system.

Here, we compared the biological activities of known TLR2 and TLR4 agonists with securinine, a novel macrophage-activating compound defined for the first time in this report. As expected, TLR4 and TLR2 agonists induced in vitro human and mouse macrophage activation and killing of phase II C. burnetii. However, the TLR4 agonist (LPS) had no impact on reducing C. burnetii infection in vivo under the conditions tested here. Variable effects were seen with the TLR2 agonist, fibroblast-stimulating lipopeptide-1 (FSL-1), and 24 h pretreatment was required to enhance resistance consistently to the bacterium. Securinine was identified in a screen of 2000 natural compounds as an activator of human macrophages, as reflected by induced cytokine production, regulation of surface adhesion molecules, and enhanced cathepsin D expression. Securinine enhanced macrophage killing of phase II C. burnetii in vitro in a TLR-independent manner and accelerated clearance of the bacterium in vivo. These results suggest that securinine or securinine-like compounds may represent novel, therapeutic adjuvants to increase innate immunity.

MATERIALS AND METHODS

Reagents and IL-8 assay
Peptidoglycan (PGN; Sigma Chemical Co., St. Louis, MO, USA), muramyl dipeptide [MDP; nucleotide-binding oligomerization domain-containing protein 2 (Nod2) agonist, Sigma Chemical Co.], Pam3CysSerLys4 (PAM3CSK4; TLR2 agonist, InvivoGen, San Diego, CA, USA), lipoteichoic acid (LTA; TLR2 agonist, Sigma Chemical Co.), Pam3CGDPKHPKSF (FSL-1, TLR2 agonist, InvivoGen), LPS (TLR4 agonist, Escherichia coli, Sigma Chemical Co.), and 2000 biologically active and structurally diverse, natural product compounds (MicroSource Discovery Systems, Gaylordsville, CT, USA) were tested on MonoMac-1 (DSM ACC 252, German Micro-organism and Cell Culture Collection, Braunschweig, Germany) or U937 (American Type Culture Collection, Manassas, VA, USA) cells for induced IL-8 production. Cellular toxicity was measured by ATP production using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

IL-8 assay
MonoMac-1 or U937 cells were cultured in complete RPMI (cRPMI) containing 10% FBS to confluency in a 96-well flat-bottom plate. Cells were then stimulated with the test compounds 20 ng/ml PMA and 0.5 µg/ml ionomycin (positive control) or PBS or DMSO/PBS (0.5%) for 24 h at 37°C and 10% CO2. TLR agonists were resuspended in PBS, whereas the MicroSource compounds were resuspended in DMSO/PBS (0.5% DMSO). After the 24-h incubation, supernatant fluid was removed and assayed for the presence of IL-8 by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Briefly, supernatant fluid of test compound-treated macrophages or IL-8 standards (2000–31.25 pg) were added to the 96-well plate with immobilized capture antibody and incubated for 2 h at room temperature. The plate was then washed four times, followed by incubation with a detection antibody for 1 h at room temperature. The plate was then washed and incubated substrate solution for 30 min. Stop solution was added, and O.D. of each well was determined by reading at 450 nm, using a wavelength correction set to 540 nm by a VERSAmax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA). O.D. values of test compounds were normalized to ng/ml using the IL-8 standard curve.

TLR activation assay
FSL-1 (2 ug/ml), LPS (1 and 0.1 ug/ml E. coli K12, InvivoGen), and securinine (50 or 25 µM) were tested on THP1-Blue-CD14 cells (InvivoGen) for TLR agonist activity, according to the manufacturer’s protocol. THP1-Blue-CD14 cells express TLR1-10 (although TLR3 and TLR9 are low), overexpress CD14, and are transfected with a reporter plasmid containing secreted embroyonic alkaline phosphatase (SEAP) under the control of a NF-{kappa}B- and AP-1-inducible promoter. TLR activation is determined by quantifying secreted SEAP. Briefly, THP1-Blue-CD14 cells at a concentration of 2 x 106 cells/ml were cultured in cRPMI containing 10% FBS in addition to glucose (4.5 µg/ml), zeocin (200 ug/ml), and blasticidin (10 µg/ml; all from InvivoGen), followed by PMA (50 ng/ml) treatment for 18 h. PMA was used to differentiate the THP1 cells to induce expression of TLR1–10. Cells were washed to remove residual PMA, and the glucose, zeocin, and blasticidin treatment was discontinued. Cells were stimulated with the compounds in cRPMI for 24 h at 37°C and 10% CO2. Supernatant fluid was removed and added to QUANTI-Blue colorimetric assay reagent for 24 h at 37°C and 10% CO2. After 24 h, samples were read at an O.D. of 655 nm by a VERSAmax tunable microplate reader (Molecular Devices). All samples were run in quadruplicate, from which means and SD were determined.

Analysis of peritoneal cells
Female Balb/c mice (6–8 weeks old) acquired from the National Cancer Institute (Frederick, MD, USA) were injected i.p. with different concentrations of FSL-1, LPS, securinine, PBS, or 0.75% DMSO in PBS for 24 h. Mice were then killed, and peritoneal fluid was recovered by injecting 10 ml HBSS into the peritoneum and extracting at least 8 ml for FACS analysis. Cells were washed, counted, and stained with mAb specific for CD11b (membrane-activated complex 1{alpha}, 10 ug/ml, BD PharMingen, Franklin Lakes, NJ, USA), Ly6C (Monts-1, 10 ug/ml; ref. [12 ]), Ly6G (RB6-8C5, 10 ug/ml; ref. [12 ]), or MHC Class II (MHC-II; AF6-120.1, 10 ug/ml, BD PharMingen). FACS analysis was performed using FACSCalibur and CellQuest software (BD PharMingen), as described previously [13 ].

Analysis of CD11b, L-selectin, and MHC-II antigen on human monocytes
Human PBMCs were collected from heparinized blood using centrifugation through Histopaque 1077 (Sigma Chemical Co.). PBMCs at the Histopaque interface were collected, plated at 1.0 x 106 cells/ml, and treated with securinine (100, 50, 25, 12.5, or 6.25 µM), FSL-1 (2 or 1 µg/ml), or 1% DMSO for 30 min or 24 h. Cells were washed and stained sequentially with anti-MHC-II (HLA-DR, -DP, and -DQ, 10 µg/ml, BD PharMingen) or anti-CD11b (MY904, 10 ug/ml, Lilly, Indianapolis, IN, USA), PE-conjugated goat anti-mouse (Jackson ImmunoResearch, West Grove, PA, USA), 10% mouse serum, and then FITC-conjugated anti-L-selectin (CD62 ligand, 10 µg/ml, BD PharMingen). FACS analysis was performed using FACSCalibur and CellQuest software (BD PharMingen), as described previously [13 ].

In vitro C. burnetii clearance
MonoMac-1, WEHI 164 (mouse cell line, ATCC), or WEHI 265 (mouse cell line, ATCC) was infected with C. burnetii [phase II Nine Mile strain, clone RSA 439; original isolate provided by Mike Minnick, Montana State University (MSU), Bozeman, MT, USA] at multiplicities of infection (MOI) of 50:1 for 24 h to allow for equal uptake of the bacterium. Cells were washed to remove all noninternalized phase II C. burnetii and stimulated with LPS (10 or 1 ug/ml), FSL-1 (10 ug/ml), PAM3CSK4 (10 ug/ml), securinine (10–25 uM), PBS, or 0.5% DMSO/PBS for 96 h. Phase II C. burnetii was purified from the cells using differential centrifugation as described by Zamboni et al. [14 ]. Briefly, cells were lysed with H2O to release phase II C. burnetii and centrifuged at 1000 g for 5 min. Supernatant fluid was collected and centrifuged at 14,000 g for 30 min to pellet the bacterium. Residual cellular debris was removed by centrifugation at 1000 g for an additional 5 min. Phase II C. burnetii was concentrated by centrifugation at 14,000 g for 30 min. Phase II C. burnetii was then subjected to a LIVE/DEAD Baclight bacterial viability and counting kit (Invitrogen, Carlsbad, CA, USA) using FACS to quantify viable C. burnetii.

In vivo phase II C. burnetii clearance studies
Female Balb/c mice (6–8 weeks old) were injected i.p. with FSL-1 (32, 16, 8, or 4 ug/mouse), LPS (100, 50, 25, or 5 ug/mouse), securinine (32 or 16 ug/mouse), PBS, or 0.75% DMSO/PBS. Mice were infected with an inoculum of 1 x 108 phase II C. burnetii i.p., 2 or 24 h after compound treatment. Mice were then killed at 24, 48, 72, or 96 h after infection, and liver, spleen, and peritoneal fluid were collected. Entire tissues were homogenized using tissue grinders, and phase II C. burnetii was purified from the cells using differential centrifugation (as described above). Twenty percent of each tissue preparation was then used for bacterial viability assays (BacLight), or bacterial DNA (genomes) was quantified by real-time PCR. For the latter, C. burnetii DNA was extracted using the UltraCleanTM microbial DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, USA). Real-time, quantitative PCR was performed using C. burnetii-specific RNA polymerase sigma factor (Rpos) primers (5'-CGCGTTCGTCAAATCCAAATA-3' and 5'-GACGCCTTCCATTTCCAAAA-3') designed with Primer Express (Applied Biosystems, Foster City, CA, USA) as described previously [15 ]. Rpos was quantified by measuring SYBR Green incorporation during real-time PCR, and PCR reactions were performed in triplicate, and data were collected using the GeneAmp 7500 sequence detection system (Applied Biosystems).

Immunofluorescence microscopy
WEHI 265 cells were plated at 5 x 105 cells/ml and treated with securinine (50 or 25 uM) or carrier/buffer control (0.5% DMSO) for 2 h, infected with phase II C. burnetii (MOI 50:1), and incubated overnight. Cytospin slide preparations of cells were fixed with 75% ETOH/25% acetone, blocked in PBS containing 10% goat serum, and stained with anti-C. burnetii [1/4000; rabbit anti-C. burnetii polyclonal serum, gift from Robert A. Heinzen, National Institutes of Health (NIH), Bethesda, MD, USA] and anti-cathepsin D, 10 µg/ml (rat anti-mouse; R&D Systems). Anti-C. burnetii was detected by addition of Alexa flour 488-conjugated goat anti-rabbit IgG (Invitrogen), and anti-cathepsin D was detected by biotin-SP-conjugated goat anti-rat IgG (Jackson ImmunoResearch), followed by the addition of Alexa flour 555-conjugated steptavidin (Invitrogen). Slides were cover-slipped using ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (Invitrogen). Cathepsin D expression was calculated by counting the number of cathepsin D-positive cells and dividing by the total number of cells x100.

Statistical analysis
One-way ANOVA followed by Tukey’s multiple comparision test was used for determining significant differences among treatment groups.

RESULTS

TLR agonist-stimulated cells kill phase II C. burnetii in vitro
In establishing a system to screen for novel innate adjuvants, we tested whether TLR stimulation of phase II C. burnetii-infected human and murine macrophage cell lines would accelerate killing of the bacterium in vitro. Preliminary assays of human macrophage cell lines showed that MonoMac-1 versus U937 cells responded more consistently and robustly to LPS (TLR4), FSL-1 (TLR2), PGN (TLR2 and/or Nod2), LTA (TLR2), and MDP (Nod2) stimulation, as measured by induced IL-8 release or NO production (data not shown). As such, MonoMac-1 cells were used for the infection assays. We first tested whether phase II C. burnetii could infect MonoMac-1 cells, and if so, what affect TLR2 (FSL-1) and TLR4 (LPS) stimulation would have on this infection. Of the different TLR2 agonists, which showed activity, FSL-1 was chosen for further study because of its efficacy and lack of toxicity in vivo [16 ]. Figure 1A shows the results of a viability assay, and as expected, based on other reports [9 , 10 ] 96 h after infection (MOI 50:1), MonoMac-1 cells contained less phase II C. burnetii if they were treated with FSL-1 or LPS versus PBS alone. This effect was not unique to human cells in that the mouse WEHI 164 macrophage cell line showed similar results (Fig. 1B) .


Figure 1
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  		Figure 1. TLR2 and TLR4 agonists induce killing of phase II C. burnetii by human and mouse macrophage cell lines. (A) Human MonoMac-1 cells infected with C. burnetii (MOI 50:1) for 24 h were treated with PBS, LPS (1 µg/ml), or FSL-1 (10 µg/ml), and the effect on the number of viable C. burnetii was compared after 96 h in culture. (B) Mouse WEHI 164 cells infected with C. burnetii for 24 h were treated with PBS, LPS (1 µg/ml), or FSL-1 (10 µg/ml), and the effect on the number of viable C. burnetii was compared after 96 h in culture using the BacLight FACS-based assay, as described in Materials and Methods. Values are means ± SD; *, P < 0.05.

FSL-1, but not LPS, variably induced increased resistance to phase II C. burnetii infection in vivo
The effect of FSL-1 and LPS on phase II C. burnetii infection in vivo was then examined. First, we confirmed activity of FSL-1 and LPS in vivo by injecting different concentrations of each agonist into the peritoneum of female Balb/c mice and then monitoring the recruitment of inflammatory leukocytes and activation of resident macrophages. As expected, each agonist induced neutrophil recruitment, as evidenced by an increase in the percentage of RB6-8C5-positive cells (>30%), and neutrophil and macrophage activation, as evidenced by increased CD11b and Ly6C expression (data not shown). To begin to examine the impact of FSL-1 and LPS on phase II C. burnetii infection, a time-course study was done in four mice treated i.p. with FSL-1 (8 ug), LPS (100 ug), or buffer alone for 2 h and then infected i.p. with 1 x 108 phase II C. burnetii. Concentrations of each agonist were based on the results obtained in the peritoneum (data not shown). At 24, 48, 72, and 96 h, the spleen phase II C. burnetii numbers (PCR quantification of bacterial genomes) were compared between the agonist-treated animals and the PBS-treated controls. The early time-points were selected to focus on innate defense responses, reasoning that time-points at 1 week and beyond would also involve considerable contributions by the adaptive immune system. As shown in Figure 2A in carrier/buffer, control-treated animals, phase II C. burnetii genomes were detected in the spleen as early as 24 h, and their numbers increased throughout the experiment. The animals showed obvious signs of disease, as reflected by ruffled fur, reduced activity, and splenomegaly (data not shown; see Fig. 2B ). Phase II C. burnetii levels were reduced in the mice treated with FSL-1 compared with carrier/buffer controls, whereas LPS pretreatment did not have an effect on phase II C. burnetii burden in this experiment (Fig. 2A) . The lack of effect of LPS may have been a result of the inhibitory effects of C. burnetii LPS for TLR4 [10 ], which for some reason, was not manifested in vitro (Fig. 1) . A second experiment was done to compare different concentrations of FSL-1 on bacterial counts and the induction of splenomegaly caused by phase II C. burnetii. As shown in Figure 2B , FSL-1 pretreatment reduced the splenomegaly associated with phase II C. burnetii infection and bacterial counts, as determined by PCR. Although detectable bacteria correlated with larger spleens, size of the spleen was not necessarily predictive of the overall burden of bacteria (Fig. 2B) . Upon additional experimentation, the effects of FSL-1 following only a 2-h pretreatment were not seen in all animals: Analysis of 18 animals treated with different concentrations of FSL-1 showed a reduction of phase II C. burnetii in only 11 mice (data not shown).


Figure 2
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  		Figure 2. Effect of TLR agonists on clearance of phase II C. burnetii in vivo. (A) Real-time PCR quantification of spleen C. burnetii DNA from single animals treated with FSL-1 (8 ug/ml), LPS (100 ug/ml), or carrier/buffer control for 2 h prior to infection with C. burnetii for 24, 48, 72, or 96 h. (B) Compares spleen weights and C. burnetii burden determined by real-time PCR from Balb/c mice injected i.p. with FSL-1 (8, 4, or 1 ug/mouse) or carrier/buffer control 2 h prior to infection with C. burnetii. Spleens were collected 96 h after infection. (C) Spleen weight, real-time PCR quantification of spleen C. burnetii genomes, and relative numbers of viable C. burnetii isolated from the spleens, as determined by the BacLight FACS-based assay from mice (n=5) treated with FSL-1 (16 ug) or carrier/buffer control for 24 h prior to infection with C. burnetii. Again, analyses were done at the 96-h postinfection time-point. *, Difference in means significant at P < 0.05.

The impact of FSL-1 on phase II C. burnetii-infected Balb/c mice was characterized further by testing whether a 24-h pretreatment might enhance the consistency of its effect. Spleen weight (Fig. 2C) and bacterial burden, as determined by PCR (Fig. 2C) , were significantly lower (more than tenfold for the bacterial counts) in five out of five mice treated with FSL-1 for 24 h prior to infection, as compared with animals treated with buffer alone. Total phase II C. burnetii genomes from the liver were lower in the FSL-1-treated mice as well (data not shown). As another test, the viable bacterial counts in the spleens of the control and FSL-1-treated animals were analyzed using the FACS-based BacLight assay used in our in vitro analyses. These results also showed that FSL-1 enhanced clearance of phase II C. burnetii (Fig. 2C) . Therefore, under the conditions tested here, FLS-1, a TLR2 agonist, enhanced innate resistance of naïve Balb/c mice to phase II C. burnetii infection, but the timing of TLR agonist treatment was important.

Securinine activate macrophages and increase phase II C. burnetii killing in vitro
In a concurrent drug discovery effort, we screened 2000 natural compounds for macrophage activation activity using IL-8 production in MonoMac-1 cells as a read-out. Securinine, a {gamma}-aminobutyric acid type A (GABAA) receptor antagonist (Fig. 3A ) [17 ], was identified as a potent inducer of IL-8 secretion in macrophages (Fig. 3B) , which has not been reported previously. As shown in Figure 3C , securinine at the same concentration used in the IL-8 assays also induced killing of phase II C. burnetii by human and mouse macrophage cell lines. To ensure this effect was not restricted to macrophage tumor cell lines, we tested the effect of securinine on primary alveolar macrophages, the cell that first encounters C. burnetii in a natural infection. Ovine alveolar macrophages were used in these experiments, as sheep, like all mammals, are susceptible to an aerosol infection by phase II C. burnetii [18 ], and their alveolar macrophages are obtained easily by lavage. As shown in Figure 3C , securinine also induced alveolar macrophage killing of phase II C. burnetii (>80%).


Figure 3
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  		Figure 3. Securinine induces IL-8 release and killing of phase II C. burnetii by macrophages. (A) The securinine structure. (B) Compares IL-8 production by MonoMac-1 cells treated with securinine (25 µM), LPS (10 µg/ml), FSL-1 (2 µg/ml), or carrier/buffer control. (C) Compares C. burnetii killing by MonoMac-1, WEHI 265, or sheep alveolar macrophage cells treated with securinine (25 µM) or carrier/buffer control (0.5% DMSO). In all infection experiments, macrophages were infected with C. burnetii (MOI 50:1) for 24 h, washed, and treated with securinine (25 µM) or carrier/buffer control (0.5% DMSO) and cultured for 96 h. The percent C. burnetii killing was determined by the following formula: 100 – (# viable C. burnetii after compound treatment/# viable C. burnetii after carrier/buffer control). *, Difference in means significant at P < 0.05.

As securinine effectively induced macrophage killing of phase II C. burnetii, we further examined its effect on macrophages. CD11b is important to the uptake of C. burnetii [19 ], and cathepsin D expression correlates with killing of the bacterium [20 ]. As such, we tested if securinine altered macrophage expression of these antigens. Concurrently, we also examined the effect of securinine on L-selectin and MHC-II antigen expression on human monocytes. Securinine up-regulated CD11b and down-regulated L-selectin expression on human blood monocytes as early as 30 min after treatment, which was similar to the expected results following FSL-1 treatment (Fig. 4 ). CD11b up-regulation and the down-regulation of L-selectin were maintained 24 h after securinine treatment, which was not seen following FSL-1 treatment (Fig. 4) . MHC-II antigen expression on 24 h securinine (6.25–50 uM samples) and FSL-1-treated cells was higher than the expression on controls (Fig. 4) . Finally, as shown in Figure 5 , cathepsin D expression was higher in securinine versus buffer control-treated phase II C. burnetii-infected cells. Thus, securinine activates macrophages, resulting in increased cytokine production, increased CD11b and MHC-II antigen expression, down-regulation of L-selectin, and increased cathepsin D expression.


Figure 4
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  		Figure 4. Securinine treatment activates human peripheral blood monocytes. Human PBMCs were treated with securinine (100, 50, 25, 12.5, or 6.25 µM), FSL-1 (2 or 1 µg/ml), or carrier/buffer control (0.5% DMSO) for 30 min or 24 h. Cells were stained with anti-MHC-II, anti-CD11b, or anti-L-selectin and analyzed by FACS. The intensity of positive cells for each of these antigens was determined by comparing with negative controls. Values are means ± SD from triplicates. *, Differences in means compared with carrier buffer/control, which are significant at a minimum P < 0.05.


Figure 5
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  		Figure 5. Securinine induces increased cathepsin D protein expression in infected macrophages. WEHI 265 cells were treated with securinine (25 µM) or carrier/buffer control (0.5% DMSO), infected with C. burnetii (MOI 50:1), and incubated overnight. Cells were then stained for cathepsin D and C. burnetii. The percentage of cathepsin D-positive cells between securinine and carrier/buffer control-treated, infected cells is compared. *, Difference in means significant at P < 0.05.

As the effects of securinine were similar to TLR agonists, we tested whether securinine may activate a TLR. These experiments were also intended to address whether the securinine preparation might be contaminated with a TLR agonist, such as LPS. Using the THP1-Blue-CD14/SEAP TLR1–10 assay, we confirmed that the activating activity of securinine (optimal concentration determined from the analyses described above) was likely not a result of TLR agonist (such as LPS) contamination or mediated by known TLRs, as TLR1, -2, -4, -5, -6, -7, and -10 signaling was not detected in the securinine preparation (Fig. 6 ). Finally, to test if securinine were directly toxic to the bacterium or the host cell, such that the bacterium could not survive, thus accounting for the results in Figure 3C , we incubated C. burnetii or the host macrophages with securinine or FSL-1 and then quantified viable bacterium or macrophages 24 h later. Neither of the compounds, at concentrations that induced killing in vitro, induced direct killing of C. burnetii or the host cells beyond the DMSO/PBS control (data not shown).


Figure 6
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  		Figure 6. Securinine does not signal through TLRs. SEAP production under the control of NF-{kappa}B (indication of TLR activation) by THP1-Blue-CD14 cells treated with LPS (0.1 µg/ml), FSL-1 (2 ug/ml), or securinine (25 µM) is shown. Values represent means ± SD from triplicate samples.

Securinine enhances clearance of phase II C. burnetii in vivo
Securinine was then tested for its effect on phase II C. burnetii infection in vivo. Balb/c mice were treated i.p. with securinine 2 h prior to infection with 1 x 108 C. burnetii. Based on the peak response to FSL-1 seen in some animals (Fig. 2) , we compared spleen weights and C. burnetii burden 96 h after challenge in the control and securinine-treated animals. As shown in Figure 7 , more than tenfold reduction in viable C. burnetii and nearly 100-fold reduction in bacterial genomes were detected in the securinine-treated compared with the control animals 96 h after infection.


Figure 7
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  		Figure 7. Securinine pretreatment increases the clearance of phase II C. burnetii in vivo. Balb/c mice (n=5) were treated i.p. with securinine (32 ug) or the carrier/buffer control. After 2 h, mice were injected i.p. with C. burnetii (1x108) and then killed 96 h later. Spleen weights and viable phase II C. burnetii counts (BacLight assay) and real-time PCR quantification of C. burnetii genomes from the spleens of five control and five securinine-treated mice are presented. Differences in means, indicated with *, are significant at P < 0.05.

DISCUSSION

Increasing innate immune responses by adjuvant therapy has been shown to be effective in enhancing resistance to infectious diseases and represents a complementary approach to vaccines and antibiotics in countering new and re-emerging infectious agents [21 ]. TLRs represent targets for most adjuvants in use today, but other innate receptors can also be targeted [22 ]. We have used high throughput screens of natural and synthetic compound libraries to identify new, innate adjuvants, which could be used in vivo, and have identified a number of novel, macrophage-specific agonists. In this study, we compared the effect of one of these, securinine, with TLR2 and TLR4 agonists, on enhancing innate resistance to phase II C. burnetii infection. As expected, TLR2- and TLR4-specific agonists induced macrophage killing of the bacterium in vitro and at least for the TLR2 agonist FSL-1, in vivo. Securinine, a GABAA receptor antagonist, also induced phase II C. burnetii killing in vitro and in vivo, which correlated with TLR-independent activation of the antimicrobial activity of the macrophage. These results suggest that securinine or securinine-like compounds may be effective adjuvants for the innate immune system.

Of the two TLR agonists tested in vivo, only FSL-1 showed efficacy in inducing enhanced clearance of phase II C. burnetii. We did not examine the basis for the lack of effect of LPS in vivo, but it may be related to the inhibitory effect of C. burnetii LPS on TLR4 [10 ], which for some reason, only manifested itself in vivo. Also, the toxicity of LPS in vivo may have masked any potential benefit. These issues are being examined currently before any definitive conclusions will be made about TLR4 agonists in this model. Although FSL-1 did work, timing of administration was important to see its effect consistently in all animals. Specifically, when the pretreatment times were increased from 2 to 24 h, the efficiency of FSL-1 in inducing enhanced phase II C. burnetii clearance went from 61% to 100% of the animals, respectively. The results with FSL-1 are consistent with previous studies showing a role for TLR2 in inducing macrophage killing of C. burnetii [9 ].

Similar to FSL-1, given 24 h prior to phase II C. burnetii infection, securinine, given only 2 h prior to challenge, enhanced clearance of the bacterium significantly at 96 h after infection, which was confirmed using two methods to measure bacterial burden (quantification of bacterial DNA and viable bacteria). The in vivo activity of securinine correlated with its capacity to activate macrophages, as evidenced by increased IL-8 production, up-regulation of CD11b, MHC-II, and cathepsin D and down-regulation of L-selectin. Current studies are looking at cathepsin D protease activity. To date, searches of the literature suggest this is the first report to demonstrate the adjuvant activity of securinine.

Securinine, a plant alkaloid, is an antagonist of the GABAA receptor [23 ], which is important in neuronal function [24 ], is expressed by peripheral monocytes, and has been shown to affect immune cell function [25 26 27 28 29 ]. It is interesting that GABA receptor agonists are thought to suppress lymphocyte cytokine production and proliferation and reactive oxygen species production by neutrophils [28 , 29 ]. Here, we show that an antagonist of the GABAA receptor drives an activating signal in macrophages. Whether securinine, though, mediates its effect through GABAA receptors awaits formal proof. It is possible that it binds other receptors on the monocyte, which mediate the responses defined here. Other plant alkaloids also activate myeloid cells via poorly defined mechanisms [30 ]. At this point, we currently have ruled out TLRs as targets for securinine. Current studies are focused on GABA receptors as well as other surface antigens, such as scavenger receptors, in ongoing efforts to define how securinine activates the macrophage.

An obvious concern with the use of an innate adjuvant in vivo as a therapeutic is induction of toxicity associated with overt inflammation and/or other indirect effects, which lead to tissue damage. However, this may not be of a concern with securinine, which does induce some neutrophil recruitment to the peritoneum after an i.p. injection, but we did not detect any obvious signs of distress in the animal. Furthermore, securinine has been used extensively in vivo for prolonged periods of time and at levels greater than the amounts used in this report with no obvious toxicity. As one example, concentrations ≥10 mg/kg given i.p. are used in rodents to achieve the neuroprotective effects of securinine without any obvious toxicity [31 ]. The adjuvant activity of securinine was seen following a single i.p. injection of 32 ug securinine, which translates to ~1.28 mg/kg, assuming a 25-g mouse. Determining the mechanism of action of securinine may reveal approaches to drive its adjuvant activity selectively and minimize its effect on the CNS even further.

These studies were not intended to determine if securinine is effective in countering Q-fever, the disease caused by highly virulent isolates of C. burnetii [8 ]. Such experiments require BSL-3-level biocontainment. However, as it has already been shown that TLR agonists [9 , 10 ] or other macrophage-activating compounds, such as chemokines [18 ] or IFN-{gamma} [19 ], induce macrophage killing of the virulent isolates of C. burnetii as well, we expect to find that securinine will have similar activity. Only a small fraction of humans, which contract virulent C. burnetii, develop debilitating disease [32 ]. Differences in how these patients’ innate immune system deals with the infection may be one factor in explaining why this occurs. If so, use of therapeutic, innate adjuvants in these patients, combined with antibiotic therapy, might be an effective treatment strategy.

In summary, we confirm predictions from the literature that TLR agonists, at least the TLR2 agonist FSL-1, induce accelerated clearance of phase II C. burnetii in vivo. We show further that the phase II Coxiella model provides a rapid screening approach for comparing the effects of multiple innate cell agonists on induced macrophage killing of an obligate intracellular pathogen. Using this model, we demonstrate for the first time that securinine, a GABAA receptor antagonist, is a macrophage-activating factor, which has similar activity on macrophages such as FSL-1. Securinine or securinine-like compounds may serve as novel immune adjuvants to increase nonspecific, innate resistance toward intracellular pathogens of macrophages, which are susceptible to induced antimicrobial activity of the leukocyte.

ACKNOWLEDGEMENTS

Funding from NIH RCE (1U54A106537-02), DoD (W9113M-04-1-0010), and NIH COBRE (5P20RR020185-02) supported this work. The efforts of J. Sentissi, S. Erickson, M. Minnick, and A. Harmsen of the MSU Coxiella Core in the production of C. burnetii used for these experiments are also acknowledged. The efforts of Drs. J. Hedges, M. Quinn, and A. Harmsen and M. Minnick and Jill Graff, who provided constructive feedback about this study and this manuscript, are appreciated.

Received April 26, 2007; revised June 27, 2007; accepted July 18, 2007.

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