Anthrax is an acute disease caused by Bacillus anthracis. Some animal species are relatively resistant to anthrax infection. This trait has been correlated to the extent of the local inflammatory reaction, suggesting innate immunity to be the first line of defense against B. anthracis infection in nonimmunized hosts. Group IIA secreted phospholipase A2 (sPLA2-IIA) is produced in particular by macrophages and possesses potent antibacterial activity especially against Gram-positive bacteria. We have previously shown in vitro that sPLA2-IIA kills both germinated B. anthracis spores and encapsulated bacilli. Here we show that sPLA2-IIA plays in vivo a protective role against experimental anthrax. Transgenic mice expressing human sPLA2-IIA are resistant to B. anthracis infection. In addition, in vivo administration of recombinant human sPLA2-IIA protects mice against B. anthracis infection. The protective effect was observed both with a highly virulent encapsulated nontoxinogenic strain and a wild-type encapsulated toxinogenic strain, showing that toxemia did not hinder the sPLA2-IIA-afforded protection. sPLA2-IIA, a natural component of the immune system, may thus be considered a novel therapeutic agent to be used in adjunct with current therapy for treating anthrax. Its anthracidal activity would be effective even against strains resistant to multiple antibiotics.

Bacillus anthracis, the etiological agent of anthrax, infects mammals (1). Upon entry into the host, the spore, the infective form of B. anthracis, germinates and gives rise to the vegetative form that produces the virulence factors, mainly the edema and lethal toxins and the capsule. Expression of these factors leads to anthrax, an association of toxemia and rapidly spreading infection evolving into septicemia with a fatal outcome in the absence of treatment. Some animal species are relatively resistant to anthrax infection. This trait has been correlated to the extent of the local inflammatory reaction (2), suggesting the occurrence of natural mechanisms controlling B. anthracis infection. Anthrax is an acute disease; therefore, innate immunity is the first line of defense that may act at an early stage of B. anthracis infection in nonimmunized hosts. However, little is known about the innate immune response that is triggered upon infection by B. anthracis spores. In a previous study, we showed that group IIA secreted phospholipase A2 (sPLA2-IIA)5 is bactericidal in vitro and ex vivo for B. anthracis as soon as it germinates and at the capsulated bacillus stage (3).

sPLA2-IIA belongs to a family of enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position, leading to the generation of lysophospholipids and free fatty acids, especially arachidonic acid (4). These products are converted into a variety of lipid mediators such as platelet-activating factor and eicosanoids, which play a major role in the initiation and modulation of inflammation (5). This production of lipid mediators is an integral component of the inflammatory reaction and might indirectly contribute to the host defense against invading pathogens. However, sPLA2-IIA plays a more direct role in the host defense reaction against bacteria; sPLA2-IIA is produced in particular by macrophages, which seems a major source of this enzyme in vivo (6, 7) and possesses potent antibacterial activity, especially toward Gram-positive bacteria (6, 8, 9, 10). The bactericidal activity always involves the hydrolysis of the phospholipids in bacterial membranes (6). The protective role of sPLA2-IIA against bacterial infections was highlighted previously in sPLA2-IIA−/− mice (11) and transgenic mice expressing human sPLA2-IIA (12). The absence of sPLA2-IIA affects the anti-bacterial response to Staphylococcus aureus infection, leading to a higher death rate compared with mice overexpressing sPLA2-IIA. Transgenic sPLA2-IIA mice are resistant to experimental infection with S. aureus and Escherichia coli (11, 13, 14). Such resistance was not correlated to a modified inflammatory status; in the steady state, sPLA2-IIA transgenic mice do not present any evidence of inflammation in the skin, liver, lung, spleen, pancreas, or thymus (12). Moreover, despite enhanced basal expression of sPLA2-IIA in these transgenic mice and in two other transgenic mouse models expressing sPLA2-IIA under inducible or specific promoters (15, 16), no evidence of an overt inflammatory status has been observed. In this latter model, in which sPLA2-IIA was overexpressed in macrophages, T cell lineages, smooth muscle cells, and fibroblasts were not significantly affected in the atherosclerotic lesions induced by high-fat diet, nor was any change in inflammatory cells observed (16).

As we had previously shown that B. anthracis is killed by sPLA2-IIA in vitro and ex vivo (3), we undertook the present study to investigate the role of sPLA2-IIA in vivo in the control of B. anthracis infection. The resistance of transgenic mice expressing human sPLA2-IIA was monitored during infection by s.c. or intranasal route with spores of 1) a septicemic nontoxinogenic B. anthracis strain to study the role on the infection per se or 2) to study a septicemic and toxinogenic B. anthracis the effect of toxemia on the afforded protection. Furthermore, we examined the efficiency of in vivo administration of recombinant sPLA2-IIA to protect naturally sPLA2-IIA-deficient mice against B. anthracis infection. Our results showed that sPLA2-IIA, a natural component of the immune system, is a major actor in host defense against B. anthracis infection and suggest that recombinant sPLA2-IIA can be considered as a novel therapeutic agent to be used adjunct to current therapy for treating anthrax.

Human sPLA2-IIA female transgenic mice (C57/BL/6NTac-TgN(sPLA2)) and C57BL/6 wild-type congenic mice (C57BL/6NTac) were obtained from Taconic Farms. In this transgenic mouse model, the transgene fragment contains the entire human sPLA2-IIA gene together with a 1.6-kb region upstream of the human sPLA2-IIA gene, with the promoter and response elements for diverse effectors (IL-6, IFN, hepatocyte NF-3, AP1, AP2, C/EBP, and cAMP response element) (12). Six- to 8-wk-old female C57BL/6 mice were purchased from Charles River Laboratories. The animals were housed in the Biosafety Level 2 and 3 animal facilities of the Institut Pasteur licensed by the French Ministry of Agriculture and complying with European regulations. The protocols used in this study were agreed by the Institut Pasteur Safety Committee, according to the standard procedures recommended by the Institut Pasteur Animal Care and Use Committee.

The B. anthracis strains used were the wild-type strain 9602, isolated from a fatal human case (17) and its ΔpagA-derivative, 9602P (18); the LD50 for both strains was <25 spores by s.c. route in OF1 mice (18) and <40 in C57BL/6 mice (this study).

Human and mouse recombinant sPLA2-IIA were produced in Drosophila S2 cells using the protocol previously described for human sPLA2-IID (19). All the sPLA2 preparations were checked for purity and protein integrity by SDS-PAGE analysis, MALDI-TOF mass spectrometry, and sPLA2 enzymatic activity assays (19). sPLA2-IIA was solubilized in PBS before injection into the ear dermis (10 μl), intranasally (30 μl) or i.v. (200 μl).

Spores were prepared as previously described (20) and stored at +4°C. Samples were diluted in PBS and injected either into the ear dermis (10 μl), into the flank (200 μl), or intranasally (30 μl). Inoculum size was verified retrospectively by plating 10-fold serial dilutions on brain heart infusion (Difco) agar plates. Survival was followed for 15 days. Bacteria in organ homogenates were counted after plating 10-fold serial dilutions on brain heart infusion agar plates.

Broncho-alveolar lavage fluids were obtained as previously described (21). Tissue homogenates were prepared as previously described (22, 23). Briefly, frozen tissues were suspended in 10 volumes of a lysis buffer containing 0.25 mM sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.5 mM PMSF, 2 μg/ml leupeptin, and 2 μg/ml aprotinin, and disrupted with an Ultra-Turrax T-25 (Janke and Kunkel) at 4°C. The homogenates were centrifuged for 5 min at 1,000 × g, and the soluble fractions were collected and centrifuged for 20 min at 20,000 × g, and the resulting supernatant was used to evaluate sPLA2 activity.

sPLA2 activity was assayed using [3H]oleate-labeled membranes of E. coli, following a modification of the method of Franson et al. (24, 25). E. coli strain CECT 101 was grown for 6–8 h at 37°C in the presence of 5 μCi/ml [3H]oleic acid (specific activity 10 Ci/mmol) until the end of the logarithmic phase. After centrifugation at 1,800 × g for 10 min at 4°C, the membranes were washed, resuspended in PBS, and autoclaved for 30–45 min. At least 95% of the radioactivity was incorporated into the phospholipid fraction. Aliquots of plasma, broncho-alveolar lavage fluid, or tissue homogenate (10–50 μl) were incubated for 15 min with 50 μl of oleate-labeled membranes (100,000–120,000 cpm) in a buffer containing 10 mM Ca2+, and the reaction was terminated by adding 100 μl of ice-cold 0.25% BSA in 100 mM Tris-HCl. After centrifugation at 2,500 × g for 15 min at 4°C, the radioactivity in the supernatants was determined by liquid scintillation counting.

The identity and level of the sPLA2-IIA protein in tissue homogenates and biological fluids were determined by inhibition of sPLA2 activity with LY311727 (Lilly Corporate Center) (22, 26).

Results are expressed as mean values ± SEM. Student’s t test and the nonparametric Wilcoxon rank sum test were used as appropriate to determine significance.

Transgenic mice expressing human sPLA2-IIA with a C57BL/6 background and wild-type C57BL/6 control mice, that do not express endogenous sPLA2-IIA because of a natural frameshift mutation in the gene encoding this enzyme (27), were inoculated into the ear with spores of the septicemic nontoxinogenic ΔpagA strain 9602P (DL50 < 40, a derivative (18) of the 9602 strain isolated from a fatal human case (17)). The bacterial load in various organs was quantified 16 h later (Fig. 1). sPLA2-IIA transgenic mice efficiently controlled B. anthracis infection locally in the ear (only 1% of the inoculated spores remained after 16 h) and at distance in the draining lymph node, spleen, liver, and blood. Significant sPLA2 enzymatic activity was detected in all these organs (Fig. 1); the enzymatic activity was fully inhibited by the sPLA2-IIA inhibitor LY311727 (data not shown). In sharp contrast, C57BL/6 wild-type mice did not control the infection and were already at the septicemic stage; they presented a high load of bacterial vegetative cells in all compartments. As expected, they did not express any detectable sPLA2-IIA activity (data not shown).

FIGURE 1.

sPLA2-IIA transgenic mice efficiently control B. anthracis infection. Transgenic mice (filled symbols) and their C57BL/6 wild-type controls (open symbols) were challenged in the ear dermis with 4 log10 (arrow) spores of the septicemic nontoxinogenic ΔpagA 9602P strain, and the bacterial load was determined 16 h later. Each symbol represents an individual animal. The results, from two independent experiments, are expressed as CFU per organ (or per milliliter for blood) for each individual mouse (n = 8). Broken lines show the threshold of detection for each compartment tested. The sPLA2 enzymatic activities were assayed in the same homogenates and were totally inhibited by the sPLA2-IIA inhibitor LY311727. No sPLA2 enzymatic activity was detected in the homogenates from the wild-type control mice.

FIGURE 1.

sPLA2-IIA transgenic mice efficiently control B. anthracis infection. Transgenic mice (filled symbols) and their C57BL/6 wild-type controls (open symbols) were challenged in the ear dermis with 4 log10 (arrow) spores of the septicemic nontoxinogenic ΔpagA 9602P strain, and the bacterial load was determined 16 h later. Each symbol represents an individual animal. The results, from two independent experiments, are expressed as CFU per organ (or per milliliter for blood) for each individual mouse (n = 8). Broken lines show the threshold of detection for each compartment tested. The sPLA2 enzymatic activities were assayed in the same homogenates and were totally inhibited by the sPLA2-IIA inhibitor LY311727. No sPLA2 enzymatic activity was detected in the homogenates from the wild-type control mice.

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In a survival assay to a lethal s.c. challenge with spores of the 9602P ΔpagA strain, all sPLA2-IIA transgenic mice survived, whereas all wild-type control mice died within 4 days (Fig. 2,a). sPLA2-IIA transgenic mice were also protected against intranasal infection with the 9602P ΔpagA strain, whereas wild-type control mice died within 3–4 days (Fig. 2 b). Significant sPLA2-IIA enzymatic activity was detected in the lung and broncho-alveolar lavage fluids of the transgenic mice (13,258 ± 1,250 and 29,453 ± 1,145 cpm/ml/min, respectively), but not in the C57BL/6 wild-type controls.

FIGURE 2.

Survival of sPLA2-IIA transgenic mice to anthrax infection. Transgenic mice (•, ▴) and their C57BL/6 wild-type controls (○, ▵) were challenged either with spores of the ΔpagA 9602P strain s.c. (a; 2.7 log10 spores; two experiments shown) or intranasally (b; 6.1 log10; two experiments shown), or s.c. (c) with 2.5 log10 (•, ○) or 3.4 log10 (▴, ▵) spores of the wild-type 9602 strain. Survival was followed for 15 days. Results of two independent experiments are shown.

FIGURE 2.

Survival of sPLA2-IIA transgenic mice to anthrax infection. Transgenic mice (•, ▴) and their C57BL/6 wild-type controls (○, ▵) were challenged either with spores of the ΔpagA 9602P strain s.c. (a; 2.7 log10 spores; two experiments shown) or intranasally (b; 6.1 log10; two experiments shown), or s.c. (c) with 2.5 log10 (•, ○) or 3.4 log10 (▴, ▵) spores of the wild-type 9602 strain. Survival was followed for 15 days. Results of two independent experiments are shown.

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In addition, we tested the ability of the sPLA2-IIA transgenic mice to withstand a s.c. infection with the fully virulent toxinogenic and encapsulated B. anthracis strain 9602 (17) (Fig. 2 c). All sPLA2-IIA transgenic mice survived the infection even with a high dose challenge, whereas all wild-type control mice died within 3 days. This finding suggests that sPLA2-IIA effectively controlled cutaneous anthrax, decreasing the bacterial load to such an extent that not enough toxin was secreted by the residual bacteria to be lethal.

To confirm the protective role of sPLA2-IIA in vivo, we tested the therapeutic effect of recombinant human sPLA2-IIA treatment on B. anthracis infection. Recombinant sPLA2-IIA of two different origins (human and mouse) was injected locally (5 μg) into sPLA2-IIA-deficient C57BL/6 mice (27) simultaneously with a s.c. challenge with spores of the septicemic nontoxinogenic ΔpagA 9602P strain; a second recombinant sPLA2-IIA local injection (5 μg) was given 6 h later. The B. anthracis load in the organs tested 16 h after bacterial inoculation was lower in all sPLA2-IIA-treated mice than in the nontreated control mice (Table I). In a survival assay, all mice treated with sPLA2-IIA at 0 and 6 h after the s.c. ΔpagA 9602P challenge survived, whereas nontreated control mice all died within 2 days (Fig. 3,a). When a single local sPLA2-IIA injection (5 μg) was performed 6 h after s.c. ΔpagA 9602P spore inoculation, 83% of the infected mice survived (Fig. 3 b).

Table I.

Human and mouse recombinant sPLA2-IIA each efficiently controls B. anthracis infectiona

Log10 CFU 9602P per Organ
Controlsh-sPLA2-IIA (5 μg)m-sPLA2-IIA (5 μg)
Ear 5.74 ± 0.06 <2.00b∗ <2.00b∗ 
Lymph node 5.60 ± 0.21 <0.60b∗ <0.60b∗ 
Liver 4.75 ± 0.72 1.94 ± 0.49d∗∗∗ <1.30c∗∗ 
Spleen 5.46 ± 0.86 1.78 ± 0.44d∗∗∗ <0.90c∗∗ 
Blood 4.68 ± 0.70 <0.60c∗∗ <0.60c∗∗ 
Log10 CFU 9602P per Organ
Controlsh-sPLA2-IIA (5 μg)m-sPLA2-IIA (5 μg)
Ear 5.74 ± 0.06 <2.00b∗ <2.00b∗ 
Lymph node 5.60 ± 0.21 <0.60b∗ <0.60b∗ 
Liver 4.75 ± 0.72 1.94 ± 0.49d∗∗∗ <1.30c∗∗ 
Spleen 5.46 ± 0.86 1.78 ± 0.44d∗∗∗ <0.90c∗∗ 
Blood 4.68 ± 0.70 <0.60c∗∗ <0.60c∗∗ 
a

sPLA2-IIA-deficient C57BL/6 mice were inoculated in the ear dermis with 3.5 log10 spores of the 9602P strain and treated locally with recombinant sPLA2-IIA of human (h-sPLA2-IIA) or murine (m-sPLA2-IIA) origin in two doses of 5 μg, one at the time of challenge and the second 6 h later. The bacterial load was determined 20 h after spore inoculation (mean ± SEM, n = 4). The thresholds are indicated in the table. Statistical significances are:

b

∗, p < 0.001;

c

∗∗, p < 0.01;

d

∗∗∗, p < 0.05.

FIGURE 3.

Administration of human or mouse recombinant sPLA2-IIA protects sPLA2-IIA-deficient C57BL/6 mice against anthrax. Mice were challenged in the ear either with: 3.2 log10 spores of the ΔpagA 9602P strain (a and b) or with 2.6 log10 spores of the wild-type 9602 strain (d). In c, the ΔpagA 9602P spore challenge (6.1 log10) was performed intranasally. Recombinant sPLA2-IIA was injected locally: a, human (h-sPLA2-IIA; ▴) or murine (m-sPLA2-IIA; •) sPLA2-IIA in two s.c. doses of 5 μg, one when challenged and the second 6 h later; b, h-sPLA2-IIA in a single s.c. dose of 5 μg at 6 h after challenge (•, ▴; two experiments shown); or c, h-sPLA2-IIA in three intranasal doses of 10 μg at 2, 6, and 30 h after challenge (•); or d, h-sPLA2-IIA in two s.c. doses of 5 μg, one when challenged and the second 6 h later (•), or in a single s.c. dose of 5 μg at 6 h after challenge (▴). Control mice were injected with PBS (○, ▵). Survival was followed for 15 days.

FIGURE 3.

Administration of human or mouse recombinant sPLA2-IIA protects sPLA2-IIA-deficient C57BL/6 mice against anthrax. Mice were challenged in the ear either with: 3.2 log10 spores of the ΔpagA 9602P strain (a and b) or with 2.6 log10 spores of the wild-type 9602 strain (d). In c, the ΔpagA 9602P spore challenge (6.1 log10) was performed intranasally. Recombinant sPLA2-IIA was injected locally: a, human (h-sPLA2-IIA; ▴) or murine (m-sPLA2-IIA; •) sPLA2-IIA in two s.c. doses of 5 μg, one when challenged and the second 6 h later; b, h-sPLA2-IIA in a single s.c. dose of 5 μg at 6 h after challenge (•, ▴; two experiments shown); or c, h-sPLA2-IIA in three intranasal doses of 10 μg at 2, 6, and 30 h after challenge (•); or d, h-sPLA2-IIA in two s.c. doses of 5 μg, one when challenged and the second 6 h later (•), or in a single s.c. dose of 5 μg at 6 h after challenge (▴). Control mice were injected with PBS (○, ▵). Survival was followed for 15 days.

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We then tested whether sPLA2-IIA could rescue mice from a lethal inhalational challenge. sPLA2-IIA-deficient C57BL/6 mice were challenged by intranasal route with spores of the septicemic nontoxinogenic ΔpagA 9602P strain and 10 μg of sPLA2-IIA was instilled intranasally at 2, 6, and 30 h after challenge. Fifty percent of the treated mice survived the lethal challenge and the observed mortality was delayed, compared with control mice that all died within 5 days (Fig. 3 c).

We then tested the potential therapeutic effect of sPLA2-IIA in extremely stringent conditions, i.e., at the late stage of infection with the ΔpagA 9602P strain, just before the onset of septicemia or when septicemia was already developing (A. Piris-Gimenez, J.-P. Corre, M. Mock, and P. L. Goossens, manuscript in preparation). sPLA2-IIA was injected locally at the site of infection (5 μg) and i.v. (20 μg) 20 h after inoculation of 9602P spores (Fig. 4). The bacterial loads in the ear and in the draining lymph node of the sPLA2-IIA-treated mice were lower than those in the nontreated controls (p < 5 × 10−3 and p = 0.025, respectively). Septicemia was controlled or delayed in a significant proportion (four of seven recipients) of the sPLA2-IIA-treated mice (p = 0.05 for the 1- and 2.5-h time points). However, the bacterial load in the ear was higher than the injected inoculum; this suggested that encapsulated vegetative cells were still present after sPLA2-IIA treatment. In a modified protocol, a higher dose of sPLA2-IIA (10 μg) was delivered locally into the ear, simultaneously with the i.v. injection (20 μg) and a survival assay was performed. No protection was observed in these conditions; however, the increase in the blood bacterial load in the first 6 h after sPLA2-IIA treatment was markedly reduced in the sPLA2-IIA-treated mice (4.8-fold ± 1.8) as compared with the nontreated mice (84-fold ± 31, p < 0.028).

FIGURE 4.

Late treatment of sPLA2-IIA-deficient C57BL/6 mice by recombinant human sPLA2-IIA leads to stabilization of B. anthracis infection. Mice were challenged in the ear with 3.2 log10 spores (arrow) of the ΔpagA 9602P strain and, 20 h later, human recombinant sPLA2-IIA was inoculated locally (5 μg into the ear) and systemically (20 μg i.v.). The bacterial load was determined in the blood before the sPLA2-IIA inoculation, 21 h after infection, and at time of euthanasia (at 22.5 h), and in the ear and the draining lymph node at 22.5 h. The results, from two independent experiments, are expressed as CFU per organ (or per milliliter for blood) for each individual mouse (n = 7). Broken lines show the threshold of detection for each compartment tested. The nonparametric Wilcoxon rank sum test was used to compare treated and nontreated mice; p < 5 × 10−3 in the ear, p = 0.025 in the draining lymph node, and p = 0.05 for the 1- and 2.5-h time points in the blood.

FIGURE 4.

Late treatment of sPLA2-IIA-deficient C57BL/6 mice by recombinant human sPLA2-IIA leads to stabilization of B. anthracis infection. Mice were challenged in the ear with 3.2 log10 spores (arrow) of the ΔpagA 9602P strain and, 20 h later, human recombinant sPLA2-IIA was inoculated locally (5 μg into the ear) and systemically (20 μg i.v.). The bacterial load was determined in the blood before the sPLA2-IIA inoculation, 21 h after infection, and at time of euthanasia (at 22.5 h), and in the ear and the draining lymph node at 22.5 h. The results, from two independent experiments, are expressed as CFU per organ (or per milliliter for blood) for each individual mouse (n = 7). Broken lines show the threshold of detection for each compartment tested. The nonparametric Wilcoxon rank sum test was used to compare treated and nontreated mice; p < 5 × 10−3 in the ear, p = 0.025 in the draining lymph node, and p = 0.05 for the 1- and 2.5-h time points in the blood.

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Finally, we tested the ability of the sPLA2-IIA treatment to protect mice against a s.c. infection with the fully virulent toxinogenic and encapsulated B. anthracis strain 9602 (17) (Fig. 3 d). All mice treated at 0 and 6 h with sPLA2-IIA, and 67% of the mice treated only at 6 h postinfection survived, whereas all wild-type control mice died within 2 days. Administration of sPLA2-IIA thus protected mice against wild-type toxinogenic and encapsulated B. anthracis strain expressing lethal toxin. This result shows that sPLA2-IIA was still able to exert its protective role even in the context of toxemia and that lethal toxin failed to interfere with the anthracidal activity of sPLA2-IIA.

Anthrax curative treatment in humans relies mainly on antibiotherapy and supportive health care (28). New therapeutic approaches are currently being developed: neutralizing mAbs against protective Ag or lethal factor (29), chemical inhibitors of edema or lethal factor (30, 31). In this study, we demonstrate that a natural component of the innate immune system significantly protected mice against B. anthracis infection: 1) transgenic mice expressing sPLA2-IIA exhibited an extremely high level of protection against cutaneous and inhalational anthrax, and 2) in vivo administration of recombinant sPLA2-IIA was efficient in controlling B. anthracis infection. In both cases, resistance was observed against infection with a highly virulent septicemic nontoxinogenic B. anthracis strain, thus showing that sPLA2-IIA controlled B. anthracis infection per se. In the s.c. murine model in which death occurs within 2 days, treatment could be postponed till 6 h after challenge. However, a single s.c. and i.v. treatment at a later stage of infection, i.e., just before the onset of septicemia or when septicemia was already developing, was inefficient in rescuing the infected mice from death. Interestingly, the local bacterial load was decreased, and septicemia could be controlled during the first hours of the sPLA2-IIA treatment, suggesting that increasing the dosage and number of sPLA2-IIA injections could help to control the infection. Protection was also observed during cutaneous and inhalational anthrax, i.e., during infection with a septicemic and toxinogenic strain, showing that sPLA2-IIA was still able to exert its protective role even in the context of toxemia.

Therefore, the bactericidal enzyme sPLA2-IIA has potential as a therapeutic agent and could be combined with the currently approved treatment of anthrax. Its action is independent of any antibiotic resistance of the B. anthracis strain causing the infection, and this is particularly valuable if the strain concerned were suspected to be multiresistant to antibiotics (as could be the case in bioterrorist threats). In support of this view, our previous studies showed that recombinant human sPLA2-IIA exerts its anthracidal effect at relatively low concentrations (ED50 ≅ 50 ng/ml) (3) and is not toxic to mammalian host cells at much higher concentrations (up to 1 μg/ml), making it suitable for therapeutic use. Indeed, at these concentrations, recombinant sPLA2-IIA neither released arachidonic acid, nor hydrolyzed membrane phospholipids of mammalian cells (19, 32, 33). In addition, sPLA2-IIA had no toxic effect on guinea pig alveolar macrophages, U937 and HL60 monocytic cell lines, rabbit platelets, or human blood monocytes as assessed by lactate dehydrogenase release and trypan blue exclusion test (Refs.34 and 35 , and L. Touqui, unpublished observations). Furthermore, intratracheal instillation of sPLA2-IIA (50 μg) has only a modest effect on surfactant phospholipid and respiratory functions in wild-type mice (21).

The remarkable high selectivity of sPLA2-IIA action against bacterial and not mammalian cells is due, in large part, to its unique substrate preference: it is specific for anionic phospholipids such as phosphatidylglycerol (19, 36, 37, 38), which is the main phospholipid component of bacterial membranes. Indeed, this phospholipid is extensively hydrolyzed in sPLA2-IIA-treated B. anthracis bacilli (3). Exogenously added sPLA2-IIA cannot hydrolyze the membrane phospholipids of mammalian cells because the outer leaflet of their plasma membrane is mainly composed by phosphatidylcholine, a very poor substrate for this enzyme (19, 32). This is because the phospholipids are asymmetrically distributed within the plasma membrane of mammalian cells (39).

sPLA2-IIA has been shown to exert in vitro a direct bactericidal effect on Gram-positive bacteria (3, 6, 8, 9, 10). In the sPLA2-IIA-transgenic mouse model used in this study, basal sPLA2-IIA mRNA expression has been observed in the liver, lung, skin, and kidney in the steady state (12); sPLA2-IIA protein has also been detected in the dermal layer of the skin and active enzyme in the serum (12). Furthermore Laine et al. (11, 13, 14) have shown that infection of these sPLA2-IIA-transgenic mice by bacteria such as S. aureus and E. coli increases sPLA2-IIA levels in the serum and mRNA expression in the liver; cytokines such as IL-1, IL-6, and TNF-α also increased sPLA2-IIA level in the serum. We believe that, in our study, sPLA2-IIA acted predominantly by a direct bactericidal effect on B. anthracis, as late inoculation of the enzyme (6 and 16 h after infection) led to a decrease of the local bacterial load; at these time points, the majority of the bacteria were encapsulated and thus extracellular. Clearly this does not exclude the potential implication of an indirect effect via modifications of the immune system per se, particularly through the interaction with a receptor on certain cell types (40). Several studies with various sPLA2s, including human group IIA, have shown that these sPLA2s can trigger iNOS induction (41), COX-2 induction (42) and IL-6, IL-8, and TNF-α cytokine secretion (43), through a mechanism involving ERK1/2 (41, 44). However, in our study, sPLA2-IIA was found effective even against infection with a B. anthracis strain secreting the toxins that are known to block the MAPK cascade (ERK/P38) through MEK cleavage (45, 46); this observation suggests a predominant direct bactericidal effect.

Administration of exogenous recombinant sPLA2-IIA is particularly relevant as B. anthracis lethal toxin inhibits the secretion of endogenous sPLA2-IIA in vitro (3), potentially decreasing the bactericidal response of the host innate immune system. The present study shows that treatment with this enzyme fully protected mice against wild-type toxinogenic B. anthracis strain expressing lethal toxin. Thus, delivery of exogenous sPLA2-IIA as an adjunct to therapy should thus overcome this potentially deleterious effect and complement any local and temporary deficiency of sPLA2-IIA, possibly a risk factor favoring the development of anthrax.

We gratefully acknowledge Agnès Fouet for helpful discussions and critical reading of the manuscript, and Catherine Le Calvez for her technical assistance in the preparation of recombinant sPLA2-IIA.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

M.P. was supported by a Fellowship Grant (PR2003-0432) from the Spanish Secretaría de Estado de Educación y Universidades.

5

Abbreviation used in this paper: sPLA2-IIA, secreted phospholipase A2 type IIA.

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