Immunostimulatory CpG Oligodeoxynucleotide Confers Protection in a Murine Model of Infection with Burkholderia pseudomallei

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhanceresistance against infection in a number of animal models, littleis known about the CpG-induced protection against acute fatalsepsis such as that associated with the highly virulent bacteriumBurkholderia pseudomallei. We previously demonstrated in anin vitro study that immunostimulatory CpG ODN 1826 enhancesphagocytosis of B. pseudomallei and induces nitric oxide synthaseand nitric oxide production by mouse macrophages. In the presentstudy, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to10 days prior to B. pseudomallei challenge conferred betterthan 90% protection. CpG ODN 1826 given 2 days before the bacterialchallenge rapidly enhanced the innate immunity of these animals,judging from the elevated serum levels of interleukin-12 (IL-12)p70and gamma interferon (IFN- ${gamma}$ ) over the baseline values. No bacteremiawas detected on day 2 in 85 to 90% of the CpG-treated animals,whereas more than 80% of the untreated animals exhibited heavybacterial loads. Although marked elevation of IFN- ${gamma}$ was foundconsistently in the infected animals 2 days after the bacterialchallenge, it was ameliorated by the CpG ODN 1826 pretreatment(P = 0.0002). Taken together, the kinetics of bacteremia andcytokine profiles presented are compatible with the possibilitythat protection by CpG ODN 1826 against acute fatal septicemicmelioidosis in this animal model is associated with a reductionof bacterial load and interference with the potential detrimentaleffect of the robust production of proinflammatory cytokinesassociated with B. pseudomallei multiplication.

INTRODUCTION

Top

Introduction

Melioidosis is a potentially fatal infectious disease endemicin many parts of the world including northern Australia, China,and several Southeast Asian countries including Thailand (3,4, 43). The disease is caused by Burkholderia pseudomallei,a facultative intracellular gram-negative bacillus found insoil and water in the areas of endemic infection. The organismis also currently considered a potential biological warfareagent (4, 14). The spectrum of infection varies from asymptomaticto acute fatal septicemia. In the northeastern part of Thailandwhere infection is endemic, melioidosis is associated with asmuch as 20% of community-acquired septicemia and relapse aftera prolonged latency period or antibiotic treatment is not infrequent(43). No vaccine is currently available for human melioidosis(43).

Patients with septicemic melioidosis and mice experimentallyinfected with B. pseudomallei have been reported to have elevatedlevels of several proinflammatory cytokines and mediators, e.g.,interleukin-6 (IL-6), IL-8, IL-12, tumor necrosis factor alpha(TNF- ${alpha}$ ), and gamma interferon-inducible protein 10 (2, 8, 22,23, 34, 37). In a murine model, exogenous gamma interferon (IFN- ${gamma}$ )could protect the animals against a subsequent fatal challengewith B. pseudomallei, and the administration of IFN- ${gamma}$ monoclonalantibody prior to the bacterial challenge significantly loweredthe 50% lethal dose (LD₅₀) and dramatically increased the bacterialburden (30). In a more recent in vitro study, the same groupof investigators showed that the ${gamma}$ -irradiated bacteria couldalso induce a rapid production of IFN- ${gamma}$ by NK cells and bystandercytotoxic CD8⁺ T lymphocytes (25). However, hyperresponsiveproduction of these mediators may also induce immunopathology(18, 28); therefore, appropriate administration of immunomodulatorsto regulate these immune aberrations would be beneficial tothe host. Unmethylated CpG oligodeoxynucleotides (CpG ODNs)are potent immunomodulators and induce highly effective protectiveimmunity in a number of chronic infectious diseases (6, 19,20). It was shown recently that the immunostimulatory CpG ODNscould also increase resistance in mice against acute nonspecificpolymicrobial sepsis (42). The CpG ODNs can also function asadjuvants to increase the effectiveness of vaccination againsta number of microbial infections (7, 19, 20). They are knownto be potent stimulators for macrophages, dendritic cells, NKcells, and some subpopulation of lymphocytes and to preferentiallyinduce Th1 development and differentiation (12, 19, 21, 33).However, no published information is currently available onprotection against acute fatal infection and severe sepsis suchas that caused by B. pseudomallei. We recently demonstratedthat CpG ODN could not only rapidly upregulate the productionof inducible nitric oxide synthase (iNOS) and NO in mouse macrophagesbut also enhance the phagocytosis of B. pseudomallei (38, 40).It is therefore logical that we should attempt to verify theeffectiveness of immunostimulatory CpG ODN against a fatal infectionin BALB/c mice challenged with B. pseudomallei. The resultsmay provide additional insights into the development of a novelapproach to enhance the innate immune response to this life-threateningdisease.

MATERIALS AND METHODS

Top

Materials and Methods

Bacterial strain and culture conditions. The strain of B. pseudomallei used in all experiments (H1038)was a recent isolate from a patient admitted to SrinagarindHospital in Khon Kaen province, where melioidosis is endemic.The patient presented clinical manifestations compatible withsepticemic melioidosis. The organism was isolated and lateridentified as an arabinose-negative biotype of B. pseudomalleiby biochemical tests, antibiotic susceptibility profile, andimmunoreactivity with polyclonal and monoclonal antibodies,as described elsewhere (1, 32, 44, 45). For experimental infectionof animals, an overnight shaken culture of B. pseudomallei inTrypticase soy broth incubated at 37°C was centrifuged,washed twice with phosphate-buffered saline (pH 7.2), resuspended,and serially diluted in phosphate-buffered saline to a desiredconcentration. The exact number of viable bacteria in the suspensionwas retrospectively determined by plating the bacteria on nutrientagar, and the colonies were counted after 30 to 48 h of incubationat 37°C and expressed as CFU. All solutions used were appropriatelysterilized, and all procedures described were carried out ina biosafety biohazard hood.

Animals. BALB/c mice, 6 to 8 weeks old (National Laboratory Animal Center,Nakorn Pathom, Thailand), were used because this inbred strainhas been shown to be highly susceptible to B. pseudomallei infection(9, 24, 37). They were housed in polypropylene cages equippedwith HEPA-filtered covers and were fed a commercial pellet dietand water ad libitum. The cages were maintained in an isolatedanimal room. All experimental procedures performed on the animalswere approved by the Animal Ethics Research Committee of theFaculty of Medicine, Khon Kaen University.

Determination of the LD₅₀. Groups of mice were inoculated intraperitoneally with differentdoses of a log-phase culture of B. pseudomallei in a 100-µlvolume. The animals were observed on a regular basis duringthe day, and the time of death was recorded. Deaths that occurredat night were assigned a time of death to coincide with themorning of observation. Under this condition, the 30-day LD₅₀for this strain as calculated by the method of Reed and Muench(29) was found to be ca. 100 CFU.

ODNs. The CpG ODN used in this study was 1826 (5' TCCATGACGTTCCTGACGTT),which was shown previously to have potent immunostimulatoryeffects on the murine immune system (12). The non-CpG ODN usedas control was 1982 (5'TCCAGGACTTCTCTCAGGTT). Both ODNs hada nuclease-resistant phosphorothioate backbone, were providedby Coley Pharmaceutical Group (Wellesley, Mass.), and containedonly a trace of endotoxin (less than 7.5 x 10^–8 endotoxinunit/ng), as determined by the Limulus assay (Whittaker Bioproducts,Walkersville, Md.). The ODN solutions were stored at 4°C.

Protocols for experimental infection and protection assay. Unless indicated otherwise, the animals were challenged on day0 by an intraperitoneal injection of either 1,000 or 5,000 CFUof a log-phase culture of B. pseudomallei H1038 in a 100-µlvolume, representing a challenge dose of 10 or 50 LD₅₀, respectively.In an initial series of experiments investigating protection,the CpG ODN was given at different times, varying between 2and 15 days relative to the time of the B. pseudomallei challenge.In brief, the animals were given an intramuscular injectionof 100 µg of either CpG ODN 1826 or non-CpG ODN 1982 ina 100-µl volume as described previously (11). After thepreliminary results were obtained, in all subsequent experimentsthe ODN injections were performed on day –2 relative tothe bacterial challenge on day 0. The animals were observeddaily as described above, and the survival data were recordedon day 30, when the experiments were terminated. Some of theanimals that remained alive on day 30 were kept under furtherobservation for as long as 3 to 4 months in order to observethe possible influence of CpG ODN 1826 on chronic infectionor a relapse, while others were sacrificed for bacterial determination.

Cytokine assays. In some experiments, mice under light anesthesia were seriallybled by retroorbital puncture, and plasma cytokine levels fromeach of these animals were individually determined by sandwichenzyme-linked immunosorbent assay using commercial kits as specifiedby the manufacturer. Depending on the experimental designs,the cytokines that were assayed included biologically activeIL-12 (IL-12p70; R&D Systems, Inc., Minneapolis, Minn.),and IFN- ${gamma}$ and TNF- ${alpha}$ (BD Pharmingen, San Diego, Calif.). The detectionlimits for these assays were 2.5 pg/ml for IL-12p70, 31.3 pg/mlfor IFN- ${gamma}$ , and 15.6 pg/ml for TNF- ${alpha}$ , but these values could bereduced further without losing much precision by using computersoftware capable of generating a four-parameter logistic curve-fit.

Determination of bacterial load in blood and tissues of infected animals. In some experiments, blood was serially collected from eachanimal via retroorbital puncture with a sterile heparinizedcapillary tube. The individual blood samples were serially diluted10-fold, 100-µl volumes were pour plated on nutrient agar,and the number of colonies, expressed as CFU, was counted after30 to 48 h of incubation at 37°C. The remaining undilutedblood was also streaked directly onto the blood agar plate andsimilarly incubated. The colonies with appearance typical ofB. pseudomallei were biochemically tested (45) and immunologicallyidentified with the latex agglutination test kit specific forthe B. pseudomallei 200-kDa exopolysaccharide antigen (1, 32).The spleen, liver, and lungs were aseptically removed from someexperimental animals to determine the numbers of residual bacteriain these organs (9). Immediately after removal, these organswere homogenized in sterile normal saline and the presence ofB. pseudomallei was determined after serial dilution, plating,and identified as described above.

Statistical analyses. For comparison of groups, the nonparametric Mann-Whitney U testor unpaired t test were applied. The difference in percent survivalbetween the CpG-treated and control groups was analyzed usingthe chi-square test. The correlation coefficient was determinedusing linear regression analysis. The level of significancefor all statistical analyses was set at P $≤$ 0.05. All data presentedas mean ± standard error of the mean (SEM) were analyzedusing Stat View version 4.5 (Abacus Concepts, Berkeley, Calif.).

RESULTS

Top

Results

Effect of CpG ODN 1826 treatment on the course of B. pseudomallei infection. In preliminary experiments, injection of BALB/c mice on day0 with challenge doses of either 1,000 CFU (10 LD₅₀) or 5,000CFU (50 LD₅₀) resulted in detectable bacteremia on day 2 (theearliest time point determined) in almost all animals. The infectedanimals died within 30 days, when the experiment was terminated.The mean time of death varied slightly from experiment to experiment,averaging between 7 and 15 days. With lower challenge doses,e.g., 100 CFU (1 LD₅₀), only a small proportion of animals developedbacteremia, but bacteria could nevertheless be detected in smallnumbers in the internal organs of all infected animals (datanot shown). Initial observations, presented in Table 1, showedthat the pretreatment of mice with CpG ODN 1826 between 2 and10 days prior to the time of bacterial challenge (day 0) conferredbetween 90 and 100% protection. However, some degree of protectioncould still be observed when the CpG ODN 1826 was given 15 daysbefore the bacterial challenge. The protection was associatedwith the CpG motif, since there was no protection in mice receivingthe control non-CpG ODN 1982 and all animals in this group died,with a mean survival time not noticeably different from thatof the untreated animals. On the other hand, when the CpG ODN1826 was given at the time of the bacterial challenge, the protectionwas not as complete, and in this case only one-third of theCpG ODN 1826-treated animals survived. Consistent with thistrend, no protection was observed when the CpG was given afterthe bacterial challenge. These limited preliminary data suggestedthat for this severe bacterial sepsis model, it takes 48 h forthe CpG protection to become effective and that the protectionwears off after 10 to 15 days. As a result of this initial observation,the remaining experiments, unless indicated otherwise, wereperformed using the 2-day pretreatment protocol.

View this table:
[in this window]
[in a new window]
TABLE 1. CpG ODN 1826 induces protective immunity against challenged with B. pseudomallei

The cumulative survival rate of mice challenged with differentdoses of B. pseudomallei was monitored for up to 30 days. Theresults showed that with a low challenge dose of 1,000 CFU (10LD₅₀), 92% of the CpG ODN 1826-treated animals survived untilday 30, when the experiments were terminated and data were analyzed.The mean survival period ± was 28.8 ± 1.25 daysfor CpG ODN 1826, 10.4 ± 2.60 days for the non-CpG ODN1982, and 6.6 ± 1.33 days for the untreated groups. Similarresults were noted when a larger challenge dose of 5,000 CFU(50 LD₅₀) was used; these values were 28.1 ± 1.35, 8.7± 1.55, and 7.1 ± 1.48 days, respectively. Thesedata demonstrated clearly that the CpG ODN 1826 treatment significantlyprolonged the survival time in B. pseudomallei-infected animals(P = 0.0001 for both). The data shown here for the severe sepsiscaused by B. pseudomallei therefore extended the protectiveefficacy of CpG ODNs reported previously for chronic infectionsby other investigators (6, 11, 19, 20).

CpG ODN 1826 treatment reduces the bacterial load in blood and averts the robust proinflammatory cytokine responses in infected animals. Because the mouse model of melioidosis described in this studyresulted in an acute disease, with most deaths occurring within7 to 10 days, the possible mechanism by which CpG ODN 1826 preventsthe rapid death in these animals needs to be examined. Bacterialloads in the day 2 blood specimens of the CpG ODN 1826-pretreatedand control (PBS-injected) B. pseudomallei-infected animalswere determined. The results presented in Fig. 1A clearly demonstratethat the bacteria could be detected in the day 2 blood in onlya small proportion of the CpG-pretreated mice (approximately13%) and that in those with bacteremia, only a small numberof bacteria (less than 400 CFU/ml of blood) were present. Inthese few bacteremic CpG-treated animals, the bacteria becameundetectable in the day 4 blood, but a subsequent breakthroughoccasionally occurred during the next few days in a few animals.This was in marked contrast to the untreated B. pseudomallei-infectedgroup (PBS control), in which the bacteremia was detected inalmost all animals (approximately 87%), some of which exhibitedextremely high blood bacterial counts (several thousand B. pseudomalleibacteria per ml of blood). Moreover, it was observed that variousnumbers of bacteria were still present in the spleen, liver,and lungs of the untreated, but not the CpG-treated, animalsthat were sacrificed on day 4, when the bacteria had alreadydisappeared from the circulation (S. Wongratanacheewin, unpublisheddata).

View larger version (9K):
[in this window]
[in a new window]
FIG. 1. Effect of CpG ODN 1826 pretreatment on bacterial load expressed as CFU (A), IFN- ${gamma}$ levels (B), and TNF- ${alpha}$ levels (C). BALB/c mice were injected with either CpG ODN 1826 ( ${circ}$ ) or PBS (•) on day –2 relative to the time of B. pseudomallei challenge (50 LD₅₀) on day 0. Blood was collected on day 2, and each specimen was used for colony count and cytokine assays. Results from individual mice (n = 15 animals per group) and group means (horizontal line) are shown. Data are representative of three independent experiments. Differences between the CpG and control groups were statistically different for panels A (P = 0.0001) and B (P = 0.0002) by the Mann-Whitney U test.

Because different lines of evidence for humans and animals suggestthe possible involvement of proinflammatory cytokines in thepathogenesis of septicemic melioidosis (22, 23, 26, 31, 34),we determined the IFN- ${gamma}$ and TNF- ${alpha}$ concentrations in the day 2blood specimens of our experimental animals (Fig. 1B and C).Without the CpG pretreatment, B. pseudomallei infection of theanimals was associated with a marked increase in the IFN- ${gamma}$ levels.In some animals, the increased level was many times higher thanthe corresponding pretreatment levels (approximately 200 pg/ml).These elevated levels were significantly attenuated in the CpGODN 1826-treated group (P = 0.0002; Fig. 1B). However, regardlessof the CpG injection status, the elevation of the TNF- ${alpha}$ levelon day 2 was not noticeably different between the two groups(Fig. 1C), but these TNF- ${alpha}$ levels were higher than the correspondingpretreatment levels in both groups (approximately 25 pg/ml).Thus, unlike IFN- ${gamma}$ , the effect of CpG ODN 1826 treatment on theTNF- ${alpha}$ levels was not very striking for the day 2 specimens. However,in a subsequent kinetic study, the effect of CpG ODN administrationon TNF- ${alpha}$ became noticeable at a later stage of infection, particularlyobvious from day 7 onward. Complete profiles of these two proinflammatorycytokines are shown in detail in Fig. 2. While the CpG ODN 1826interfered with the robust IFN- ${gamma}$ response early during the courseof infection (days 2 and 4 in Fig. 2A), its effect on the TNF- ${alpha}$ response was noted only during the later stage of infection(Fig. 2B). Within 1 week of infection, the TNF- ${alpha}$ levels in theCpG-treated group had already returned to levels approachingthat of the baseline, while the levels in animals without theCpG treatment remained significantly elevated until the animalsdied. However, in this study we did not observe any correlationbetween the time of death and the bacterial load or the cytokinepeak (both the magnitude and the time). Nevertheless, in someanimals, a low degree of correlation with the level of TNF- ${alpha}$ was noted.

View larger version (20K):
[in this window]
[in a new window]
FIG. 2. Effects of CpG ODN 1826 pretreatment on IFN- ${gamma}$ (A) and TNF- ${alpha}$ (B) serum profiles of B. pseudomallei-infected animals. Animals were treated with CpG ODN 1826 and challenged with B. pseudomallei (50 LD₅₀) as described in the legend to Fig. 1. The animals in each group (n = 15) were bled serially as shown (the numbers of specimens at each time point were not equal since some animals in the control group died before day 15). Differences between groups at each time point were calculated by the Mann-Whitney U test, and those indicated by * were statistically significant (P = 0.0002 and P = 0.0047 for IFN- ${gamma}$ on days 2 and 4, respectively, and P = 0.032, P = 0.0058, and P = 0.0087 for TNF- ${alpha}$ on days 7, 9, and 15, respectively). Error bars represent SEM.

CpG ODN 1826 pretreatment induces early innate cytokine responses in mice prior to the time of bacterial challenge. The obligatory role of IFN- ${gamma}$ for host survival has been demonstratedin a murine model of B. pseudomallei infection (30). However,the current data (presented above) clearly demonstrated thatthe elevated levels of IFN- ${gamma}$ detected in the day 2 specimensin this highly susceptible BALB/c mouse model were not sufficientto protect them from acute lethal melioidosis. One of the protectivemechanisms against this bacterial infection is attributableto the ability of the macrophages to produce NO (27), and werecently showed that CpG ODN 1826 could upregulate iNOS andNO production (38). It would be interesting to determine ifthe pretreatment of mice with CpG ODN 1826, which provided significantprotection against a fatal challenge 2 days later, is also associatedwith the early rise in the levels of IFN- ${gamma}$ -inducing cytokines,such as the biologically active form of IL-12 (IL-12p70) andIL-18. These proinflammatory cytokines may then stimulate theproduction of IFN- ${gamma}$ and activate other effectors of innate immunityto a level sufficient to control bacterial multiplication. Resultspresented in Fig. 3 are compatible with our expectation, sincethe levels of both the IFN- ${gamma}$ -inducing cytokine (i.e., IL-12p70)and IFN- ${gamma}$ in the circulation were found to be significantly elevated(P = 0.0001) within 24 h of the CpG ODN 1826 injection (day–1 in Fig. 3). Whether this small but highly significantearly rise in the levels of IL-12p70 and IFN- ${gamma}$ by itself wassufficient to control the growth and survival of the bacteriato a more tolerable level or whether another mechanism, e.g.,interference with subsequent bacterium-induced excessive anduncontrolled proinflammatory cytokine production, may be involvedremains to be determined. It should be mentioned that we previouslypresented in vitro evidence indicating that the B. pseudomallei-killingactivity of macrophages could be enhanced by pretreatment ofthese cells with IFN- ${gamma}$ (38, 40). Moreover, we have limited data(unpublished) showing that adherent spleen cells from CpG-pretreatedmice had enhanced iNOS production and microbicidal activityagainst B. pseudomallei compared with those from untreated controlanimals.

View larger version (23K):
[in this window]
[in a new window]
FIG.3. Innate cytokine profiles in the serum of infected BALB/c mice with or without a prior CpG ODN 1826 pretreatment. The animals were injected with 100 µg of CpG ODN 1826 on day –2 (arrow) relative to the challenge on day 0 (arrow) with B. pseudomallei (50 LD₅₀), as described in the legend to Fig. 1. The animals were bled serially on day –5, day –1 (1 day after either CpG ODN 1826 or PBS injection), day 1 (1 day after the B. pseudomallei challenge), day 2, day 6, and day 7. It should be noted that compared with day –5 (**), CpG ODN 1826 induced a significant elevation of both IL-12p70 (A) and IFN- ${gamma}$ (B) cytokines on day –1 (P = 0.0001 for both by the unpaired t test). The CpG ODN treatment was associated with a marked attenuation of the IFN- ${gamma}$ (P = 0.0002) and, to a lesser extent, IL-12p70 (P = 0.0486) production on day 2 (*) following a bacterial challenge. The number at the bottom of each bar indicates the number of specimens available for analysis at each time point. Data are representative results of two independent experiments. Error bars indicate SEM.

DISCUSSION

Top

Discussion

B. pseudomallei infection exhibits broad clinical manifestationsin humans and animals. The mechanisms underlying the developmentof different forms of disease remain poorly understood, butthere is no doubt that the degree of virulence of the organismsand host factors are important in pathogenesis and disease progression(4, 9, 24-27, 30, 36, 37). The results of the present studyreveal new information not only about the more complete bacteriallyinduced proinflammatory-cytokine profiles and development ofbacteremia in this animal model of melioidosis sepsis but alsoabout the way in which these could be averted by appropriateadministration of an immunomodulating agent. The levels of anumber of proinflammatory cytokines and other mediators havebeen reported to be elevated, and this has been correlated tosome extent with disease severity in patients with severe melioidosis(2, 8, 22, 23, 34). However, some of these cytokines, particularlyIFN- ${gamma}$ and, to a lesser extent, TNF- ${alpha}$ and IL-12p70, have been shownto provide protection against melioidosis in experimental animals(30). On the other hand, it is well documented that excessiveand unregulated production of proinflammatory cytokines maybe detrimental to the host and can result in septic shock anddeath, depending on the bacterial burden and time of cytokineproduction relative to the time of bacterial exposure (13, 18,28). The data presented in this study showed for the first timethat without CpG intervention, practically all animals diedand that the time of death for each animal correlated with neitherthe time of development and magnitude of bacteremia nor thepeak levels of cytokines. Some untreated B. pseudomallei-infected(control) animals with heavy bacterial loads and extremely highIFN- ${gamma}$ levels survived for several more days without clinicalsigns of infection when the bacteria had already disappearedfrom the blood and the elevated level of IFN- ${gamma}$ had returned toa baseline value. However, almost all of these animals died,mostly before day 30, when the experiment was terminated. Inthe experiment whose results are shown in Fig. 1 and 2, thepresence of bacteria in the internal organs was not determined.However, we have limited data from a parallel experiment demonstratingthat B. pseudomallei could still be detected in the spleen,liver, and lungs of the untreated animals, but not the CpG-treatedanimals, that were sacrificed on day 4 when the bacteria hadalready disappeared from the circulation (Wongratanacheewin,unpublished).

The CpG ODN 1826 given to the animals 2 days before the bacterialchallenge dramatically reduced bacteremia, prolonged the survivaltime, and conferred to these animals a significant degree ofprotection against lethal sepsis. No bacteremia could be detectedin a majority of CpG-treated animals (Fig. 1A), and in the fewanimals in which bacteremia was detected, the bacterial loadwas considerably smaller than what was normally found in untreatedanimals. It appears, therefore, that CpG could not only favorablyaffect the outcome of acute infection, as shown here, but couldalso reduce the chance of the infection progressing to a chronicphase, since the CpG-treated animals sacrificed after day 30had no demonstrable B. pseudomallei in their internal organs(data not shown). Interference with bacterial multiplicationand prolongation of the survival time in the CpG-treated groupmust be related to enhancing innate immunity immediately priorto the time of bacterial challenge (Fig. 3), since little orno protection was observed when the same quantity of CpG ODN1826 was given at the same time as or after the bacterial challenge(Table 1). The rapid induction of the IFN- ${gamma}$ -inducing cytokineIL-12p70 by CpG ODN, which induced IFN- ${gamma}$ immediately thereafter,would preactivate the effector cells of innate immunity, thusenhancing their antimicrobial power at the time of bacterialchallenge 2 days later. Although an array of other cytokines,including IL-18 and IL-27, are known to synergistically playa role in inducing IFN- ${gamma}$ production (5, 35), in the present studyonly the IFN- ${gamma}$ -inducing cytokine IL-12p70 was analyzed, becauseit appears to act upstream in the induction of IFN- ${gamma}$ synthesis.The protective role of an early IFN- ${gamma}$ response in a murine melioidosismodel had been demonstrated using other approaches, e.g., neutralizingmonoclonal antibody to IFN- ${gamma}$ and IFN- ${gamma}$ receptor knockout animals(30). We previously demonstrated in an in vitro study that theCpG ODN 1826 not only could upregulate the expression and productionof iNOS and NO but also could enhance the uptake of B. pseudomallei(38, 40) by mouse macrophages. These in vitro data are in accordwith our more recent in vivo observations that the adherentspleen cells of mice injected with CpG 2 days earlier couldupregulate the production of iNOS and enhance both the uptakeand intracellular killing of B. pseudomallei (data not shown).However, the possibility that the CpG might also enhance killingby neutrophils cannot be ruled out, since it was demonstratedpreviously that the CpG could increase phagocytosis and productionof reactive oxygen intermediates of the neutrophils (42).

It is possible that in the absence of CpG ODN 1826, the B. pseudomalleiused in our model is allowed to replicate rapidly and unopposedto a level that cannot be successfully controlled by the IFN- ${gamma}$ that is subsequently induced by the bacteria. This speculationis consistent with our previous findings that, compared withother gram-negative bacteria, not only was B. pseudomallei inherentlyless potent in stimulating the effectors of innate immunity,e.g., TNF- ${alpha}$ , IFN-ß, iNOS, and NO, but also it did sowith slower kinetics (39, 41). The robust production of IFN- ${gamma}$ noted on day 2 in the untreated B. pseudomallei-infected animalswas most probably related to the presence of a very large bacterialburden, and we theorize that it was then too late to controlthe ongoing infection. At this time, the macrophages might alsobe so severely damaged that they are unable to mount an effectiveantimicrobial response against the bacteria. It has been observedthat patients with septicemic melioidosis not only have higherblood bacterial counts but also have a higher mortality ratethan those infected with other gram-negative bacteria (31).It should be mentioned that both the bacterial load and theproinflammatory cytokine levels noted in the present study wereconsiderably greater than those found in the patients. It isunlikely, however, that in our murine model, the transient elevationof the IFN- ${gamma}$ level in the untreated B. pseudomallei-infectedanimals by itself (Fig. 2 and 3) is the primary cause of death,since some of the animals survived for many more days afterthe IFN- ${gamma}$ level returned to normal. It is possible that otherfactors, e.g., TNF- ${alpha}$ , might contribute to fatal sepsis in thisanimal model. Our data on TNF- ${alpha}$ suggested that the elevated levelof this cytokine during the late stage of infection (e.g., fromday 7 onward) might be more closely related to the outcome ofinfection. For example, the results presented in Fig. 2 showedthat from day 7 onward, the TNF- ${alpha}$ levels in the untreated, B.pseudomallei-infected mice remained elevated until the timeof death compared with the levels in the CpG-treated animals.This was particularly obvious on days 9 (P = 0.0058) and 15(P = 0.0087), when the difference between the two groups becamehighly statistically significant. In accord with this observation,it was shown previously that the more resistant C57BL/6 miceexhibited a considerably lower level of TNF- ${alpha}$ than did the moresusceptible BALB/c mice, which succumbed to fatal sepsis followingB. pseudomallei infection (26). These observations support theprevious results of studies with humans, showing that the levelsof TNF- ${alpha}$ in melioidosis patients correlated with the severityof the disease and could be considered a predictive marker forthe outcome of infection (22, 34). Since B. pseudomallei itselfis a poor stimulator of the innate immune response (39, 41),it is possible that the initially weak innate immune responseallows a rapid multiplication of the bacteria, resulting inan excessive and uncontrolled cytokine production, which couldlead to shock and death. Taken together, these data are compatiblewith the notion that the enhanced innate immune response inducedby CpG administration would avert this process. On the otherhand, one should also keep in mind the possible direct actionof CpG ODN 1826 administration in ameliorating the potentialdetrimental effects of a subsequent bacterially induced cytokineproduction, similar to the endotoxin tolerance which is knownto protect the host against infections (15-17, 46). In the presentstudy, we found no direct evidence that CpG ODN 1826 could negativelyregulate the proinflammatory cytokine response in this experimentalmodel of melioidosis. However, it is a question that shouldreceive further attention, particularly in view of the morerecent report showing that CpG differed from lipopolysaccharidein inducing IFN- ${gamma}$ production (10, 18).

Taken together, the data presented here suggest that a rapidinduction of innate cytokines by CpG treatment before the timeof bacterial challenge would determine whether the disease isobligatory and will proceed to an acute lethal outcome or tofull recovery without going through a chronic stage or latencyphase. We feel that the time when the proinflammatory cytokinesare induced by immunomodulating agents is highly critical, andthis window is rather narrow in melioidosis, requiring at least48 h prior to the infectious challenge but lasting only 10 to15 days. It should be interesting to determine whether the protectivewindow for this potentially fatal acute infection could be increasedif a larger dose of CpG is used. Our proposal based on the B.pseudomallei model presented in this communication is in accordwith the prediction of the occurrence of sepsis and the outcomeof infection proposed previously by other investigators (18,28).

In conclusion, the results presented in this study showed thatCpG ODN 1826 is a potentially useful immunomodulating agentthat is as effective in preventing an acute and lethal sepsisin animals experimentally infected with B. pseudomallei as itis in preventing chronic infections in other experimental models.It provides new insights for future investigation if one isto develop a novel approach to the management of patients withsevere melioidosis. Lastly, it should be mentioned that in theabsence of vaccine and in the setting of ineffective antibiotictherapy and the potential danger of B. pseudomallei as a biologicalwarfare agent, the beneficial effect offered by immunomodulatingagent such as CpG ODN should provide an alternative approachto preventing fatal sepsis.

ACKNOWLEDGMENTS

This work was supported by Thailand Research Fund (RDG 4530209)and Chulabhorn Research Institute, Bangkok, Thailand.

We appreciate the kind comments and suggestions from NicholusJ. White (University of Oxford, Oxford, United Kingdom). Wealso thank Bryan R. Hamman for assistance during the preparationof the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Phone: (66-43) 363515. Fax: (66-43) 348385. E-mail, sura_wng{at}kku.ac.th.

Editor: J. D. Clements

REFERENCES

Top