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Infection and Immunity, July 1999, p. 3593-3600, Vol. 67, No. 7
Department of Infectious and Tropical
Diseases,
Received 29 June 1998/Returned for modification 13 August
1998/Accepted 7 April 1999
Burkholderia pseudomallei, the causative agent of
melioidosis, is a gram-negative bacterium capable of causing either
acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection
in areas where it is endemic, there is essentially no information on
the mechanisms of protective immunity to the bacterium. We describe
here a murine model of either acute or chronic infection with B. pseudomallei in Taylor Outbred (TO) mice which mimics many
features of the human pathology. Intraperitoneal infection of TO mice
at doses of >106 CFU resulted in acute septic shock and
death within 2 days. In contrast, at lower doses mice were able to
clear the inoculum from the liver and spleen over a 3- to 4-week
period, but persistence of the organism at other sites resulted in a
chronic infection of between 2 and 16 months duration which was
eventually lethal in all of the animals tested. Resistance to acute
infection with B. pseudomallei was absolutely dependent
upon the production of gamma interferon (IFN- Melioidosis is a life-threatening
infectious disease caused by the gram-negative bacterium
Burkholderia (formerly Pseudomonas) pseudomallei. The organism is endemic in Southeast Asia and
Northern Australia and can be isolated from a variety of environmental sources, especially soil, ponds, and rice paddies (8). The disease may well be underreported in other areas of the tropics. Infection occurs in humans and other animals. Natural infection is
thought to occur via either skin trauma or inhalation of contaminated aerosols (19). Melioidosis is a major cause of mortality in the Northeast of Thailand, where it accounts for approximately 40% of
lethal, community-acquired septicemia (5). B. pseudomallei can cause infection in any organ, although pathology
occurs mainly in the lungs, spleen, and liver (24, 36). A
striking feature of melioidosis is the extremely varied spectrum of
clinical presentation. In many cases, infection presents as an acute
illness characterized by septic shock. In contrast, some individuals
become latently infected with no obvious signs of disease. However,
after impairment of immune defenses, such as is seen in diabetes
mellitus, renal failure, and steroid treatment, the organism can be
reactivated and cause acute lethal sepsis. In fact, relapse of
asymptomatic infection has been reported over 20 years after initial
exposure to the organism, suggesting an ability to evade immune
defenses (18, 20). The ability of the bacterium to cause a
long-term latent infection suggests that B. pseudomallei is
capable of surviving in an intracellular environment. Results obtained
with cell culture systems support this view, in that virulent B. pseudomallei is able to infect and grow within various cell lines
as well as human polymorphonuclear leukocytes (PMNL) and monocytes
(11, 16, 25). However, neither the site of latency, the
mechanisms by which the organism avoids the bactericidal effects of the
host immune response, nor the pathways of protective immunity
against this bacterium have been identified.
In many other intracellular bacterial infections, the rapid production
of proinflammatory and phagocyte-activating cytokines is a key
determinant of resistance of the host. The production of gamma
interferon (IFN- The purpose of this study was to establish a murine model of
melioidosis in order to examine the relative importance of IFN- Bacteria.
A gentamicin-sensitive strain (708a) of B. pseudomallei, originally obtained from the splenic abscess of a
melioidosis patient, was used in all experiments. This strain was
isolated from Sappasitprasong Hospital, Ubon Ratchatani, Thailand.
Bacteria were subcultured on Columbia agar (Oxoid) and incubated for
24 h at 37°C. Several young colonies were scraped from the agar
and suspended in sterile pyrogen-free saline solution (PFS). The
suspension was adjusted to an absorbance (optical density) of 0.50 at a
600-nm wavelength. A frozen stock was prepared at a concentration of
108 CFU/ml in PFS with 10% glycerol (vol/vol) and stored
at Animal model of infection.
Female, Taylor Outbred (TO) mice
(Tuck) or BALB/c mice (Harlan Olac) were used at 10 to 12 weeks of age.
In other experiments, mice with a targeted gene deletion of the IFN- Quantitation of bacterial load and histology.
Small sections
of spleen and liver from infected mice were taken immediately after the
organs had been harvested and placed in 2% neutral buffered formalin
for at least 24 h. This procedure fixed and disinfected the
tissues, which were then processed for routine histological examination
by using 5-µm-thick paraffin-embedded sections stained with
hematoxylin and eosin. The remainder of the organs were placed into 5 ml of PFS containing 0.5% (vol/vol) Triton X-100 (BDH) and disrupted
by passing through a 100-µm-pore-size nylon mesh sieve (Falcon).
Serial 10-fold dilutions of the resulting cell suspensions were spread
in duplicate onto Columbia agar, and colonies were counted after
48 h incubation at 37°C. The remainder of the cell suspensions
were added into 10 ml of brain heart infusion broth (Oxoid) and
incubated at 37°C for up to 1 week. The positive broths were
subcultured onto Columbia agar, and the bacteria were identified. All
negative broths were subcultured on day 7 before being discarded.
Neutralizing anti-cytokine antibodies.
To neutralize
specific cytokines in vivo, mice were injected i.p. at the time of
infection with either 300 µg of hamster anti-mouse IFN- Statistics.
Statistics analysis (unpaired t test
and Kaplan-Meier survival analysis) were performed by using GraphPad
Prism software (GraphPad, San Diego, Calif.). A P value of
<0.05 was considered significant.
Comparison of susceptibility of BALB/c versus TO mice to B. pseudomallei.
In order to establish a model of melioidosis which
would encompass both acute and chronic features of the human disease,
we compared the course of infection in inbred BALB/c mice versus the
outbred mouse strain TO. Mice were injected i.p. with various doses of
B. pseudomallei 708a, and cumulative survival was monitored up to a period of 65 days postinfection. Infection of TO mice resulted
in death within 48 h only at a dose of 107 CFU per
mouse and delayed mortality between days 25 to 40 at 106
CFU (Fig. 1). Lower doses of bacteria
generally resulted in chronic infections, after which death of
remaining mice occurred in proportion to the initial inoculating dose
of bacteria, i.e., mice receiving the lower dose lived longer. In
contrast, acute lethal sepsis occurred within 3 days in all BALB/c mice
given 107, 106, or 105 CFU of
B. pseudomallei, whereas lower doses were sublethal within the first 2 months of infection (data not shown). In view of their greater resistance, TO mice were subsequently used in all further in
vivo experiments.
Time course of bacterial clearance after infection with B. pseudomallei.
To investigate the kinetics of bacterial replication
in the tissues, TO mice were infected with a sublethal dose (2 × 105 CFU) of B. pseudomallei, and the quantity of
bacteria in the spleen and liver was determined at various time
intervals. At the earliest time point examined (24 h postinfection),
the numbers of bacteria in each organ approximated that in the original
infecting dose (Fig. 2). However, the
splenic load of bacteria progressively decreased over the following 4 days such that by day 8, two of five mice contained only low numbers of
bacteria as demonstrated by viable counts, two of five mice were
negative on the basis of direct plating of tissue homogenates but
showed positive growth when the homogenates were cultured in enrichment
broth, and one of the five animals was found to be sterile by both
culture criteria. From one experiment, a single animal which had shown
no clinical sign of illness was examined, and multiple abscesses were
found in the spleen yielding more than 108 CFU per organ,
suggesting that in some cases focal growth of the organism can occur
without overt signs of infection. Splenomegaly was a prominent feature
of the infection and occurred by day 4 and increased progressively with
time in contrast to the bacterial burden, which decreased over this
period (data not shown). Histopathological examination of the spleens
of sublethally infected mice showed an initial infiltration of
neutrophils followed by macrophages and lymphocytes (Fig.
3). A similar pattern of progressive
reduction of bacterial loads was observed in the liver; however, the
clearance of bacteria from this organ was slightly slower than from the spleen (Fig. 2B). Histology sections of the lung showed minimal involvement of this organ by this route of infection.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Obligatory Role of Gamma Interferon for Host
Survival in a Murine Model of Infection with Burkholderia
pseudomallei
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) in vivo.
Administration of neutralizing monoclonal antibody against IFN-
lowered the 50% lethal dose from >5 × 105 to ca. 2 CFU and was associated with 8,500- and 4,400-fold increases in the
bacterial burdens in the liver and spleen, respectively, together with
extensive destruction of lymphoid architecture in the latter organ
within 48 h. Neutralization of either tumor necrosis factor alpha
or interleukin-12 but not granulocyte-macrophage colony-stimulating
factor, also increased susceptibility to infection in vivo. Together,
these results provide the first evidence of a host protective mechanism
against B. pseudomallei. The rapid production of IFN-
within the first day of infection determines whether the infection
proceeds to an acute lethal outcome or becomes chronic.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) is of particular importance in controlling the
rate of bacterial growth, as shown by the extreme susceptibility to
intracellular pathogens of mice depleted of IFN- by either gene
deletion or neutralizing monoclonal antibody (MAb) (7, 23).
However, in the context of gram-negative infection, IFN- has also
been associated with the development of septic shock, suggesting that
in the presence of lipopolysaccharide, IFN- can be either beneficial
or detrimental depending on the bacterial burden and time of production
(1, 4, 15). Other cytokines, including tumor necrosis factor
alpha (TNF-
), interleukin-12 (IL-12), and IL-6 may also confer
protection or cause pathology, depending on the experimental system.
The role of these cytokines in protective immunity against infection
with B. pseudomallei has until now not been examined in an
experimental model, although serum levels of TNF-, IFN-, and
soluble IL-2 receptors, as well as IL-6 and IL-8, are elevated and
correlated with disease severity in patients with severe melioidosis,
providing predictive markers of outcome (3, 13, 29).
and
other proinflammatory cytokines in host resistance. Previous attempts
to develop animal models of this disease have used a range of species,
including ferrets, guinea pigs, hamsters and, to a limited extent,
inbred mice (9, 21, 34, 35). In many cases, infection with
relatively low numbers of B. pseudomallei has invariably led
to acute lethal sepsis, making it difficult to dissect the mechanism of
protection. Generation of models of chronic disease have been reported
in some species, but the immunological basis of latency has not been
examined (21). We now present evidence that infection of
outbred TO mice provides a model of both acute and chronic melioidosis,
depending upon the initial inoculum. We report here that, in
particular, IFN-, as well as IL-12 and TNF-, are essential
mediators of protective immunity. These data provide the first evidence
for the mechanism of immune control of this clinically important pathogen.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C. When required to inoculate into mice, the bacteria were
thawed, washed once with PFS, and diluted to the required concentration in PFS. Identification of the strain both in the initial inoculum and
when isolated from acute and chronic infections was confirmed by API
20NE kits (BioMérieux), and a standard disc diffusion method was
used to determine susceptibility to gentamicin.
receptor (G129) were compared with their wild type (129SVEV) as
controls. These were originally obtained from Bantin & Kingman (Hull,
United Kingdom) and bred at the London School of Hygiene and Tropical
Medicine (LSHTM) under aseptic conditions. All animals were maintained with access to food and water ad libitum under Animal Biohazard Containment Level 3 conditions throughout the experiments. Mice were
infected intraperitoneally (i.p.) or subcutaneously with various
concentrations of viable B. pseudomallei in 0.2 ml of PFS.
All infections were performed in a class I Biosafety cabinet, and
infected animals were maintained within a negative-pressure, HEPA-filtered flexible film isolator (MDH).
MAb (H22)
(provided by R. D. Schreiber, Washington University, St. Louis,
Mo.), 300 µg of hamster anti-mouse TNF MAb (TN319.12), 1 mg of rat
anti-mouse IL-12 MAb (C17.8) (provided by C. Engwerda, Department of
Infectious and Tropical Diseases, LSHTM, and originally obtained from
G. Trinchieri, Wistar Institute), or 300 µg of rat anti-mouse
granulocyte-macrophage colony-stimulating factor (GM-CSF) MAb
(MP1-22E9) diluted in PFS. Control mice were injected with appropriate
concentrations of either PFS, hamster isotype control antibody (L2), or
purified rat immunoglobulin G (IgG; Sigma, Poole, Dorset, United Kingdom).
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (18K):
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FIG. 1.
Effects of B. pseudomallei on the mortality
of TO mice. Mice were infected i.p. with doses of B. pseudomallei from 107 ( 
), 106 (
),
105 (
), 104
(
), or
103
(
) CFU
per mouse versus use of PFS alone (
). Animals were observed daily,
and the percent survival values were plotted against time. Infection
with either 107 or 106 CFU of the bacteria (but
not lower doses) resulted in a significantly earlier death compared to
uninfected animals (P <0.02).
View larger version (13K):
[in a new window]
FIG. 2.
Kinetics of B. pseudomallei clearance from
the livers and spleens of infected mice. Mice were infected i.p. with
105 B. pseudomallei, and at various times
bacterial loads were determined in the spleen (A) or liver (B) as
described in Materials and Methods. Data are presented as the CFU per
organ for five individual mice per group. An asterisk represents a
single animal with splenic abscesses.
View larger version (125K):
[in a new window]
FIG. 3.
Histology of infection with B. pseudomallei
in the spleen after neutralization of IFN- in vivo. Mice infected
i.p. with B. pseudomallei and treated with either an isotype
control antibody L2 (a and b) or anti-IFN- MAb H22 (c and d) at
40 h postinfection. Evidence of destruction of the red pulp (arrow
in panel c) and disruption of normal lymphoid architecture (arrow in
panel d). (e) High-power view of infected, H22-treated mice showing
large numbers of the bacteria (arrows). Each photograph represents a
typical view of the entire section and for all mice studied (three mice
per group).
Role of IFN- in resistance to acute infection with B. pseudomallei.
IFN- has been implicated in both protective
immunity and the pathology associated with infection with gram-negative
bacteria. To determine the contribution of IFN- in resistance to
B. pseudomallei, mice were treated with either saline or the
neutralizing hamster anti-mouse IFN- MAb (H22) immediately prior to
infection with a sublethal dose of viable bacteria, and mortality was
monitored. Saline-treated, infected mice survived for the duration of
the experiment without any clinical signs of infection. In contrast, mice treated with H22 MAb showed visible signs of acute sepsis (i.e.,
weight loss and lethargy) within 20 h of infection, and all were
dead by 40 h after infection (Fig.
4). Similar conversion to an acutely
lethal event was also observed if neutralization of IFN- was delayed
until 7 days postinfection, although the mortality kinetics observed in
this group indicated that death was slightly delayed. All animals died
within 5 days of antibody administration, whereas all control infected
mice survived until the termination of the experiment on day 30 (data
not shown). Treatment of infected mice with an isotype-matched control
hamster IgG (L2) resulted in an identical outcome to the treatment with saline plus B. pseudomallei, whereas mice treated with H22
alone and not infected showed no illness at any point. In contrast to the dramatic effects of neutralizing IFN-, MAb to murine GM-CSF at
concentrations known to inhibit resistance to other pathogens, including Cryptococcus neoformans, Legionella
pneumophila, and Candida albicans (6, 37),
had no effect on the outcome of infection (Fig. 4).
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); anti-IFN-
), or 
) at 300 µg/mouse at the time of
i.p. infection with B. pseudomallei (2 × 105 CFU). Results are presented as the percent survival
against time. Where multiple groups all showed 100% survival, data
points have been separated for clarity of presentation. Infected mice
treated with H22 resulted in a significantly earlier death compared to
B. pseudomallei plus control antibodies (P < 0.02). The results are representative of two independent
experiments.
depletion, the livers and
spleens of infected mice were harvested to quantitate the number of
bacteria per organ. Respectively, 4,400- and 8,500-fold more bacteria
were isolated from the spleen and liver of anti-IFN- MAb-treated,
infected mice than from the control group (Fig.
5). Although the bacterial loads in both
liver and spleen from the IFN--depleted mice were similarly high,
the histopathology of these two organs was distinctive. In the spleen,
lymphoid architecture, particularly in the red pulp, was extensively
destroyed, and large numbers of bacteria were spread throughout the
organ and easily observed on hematoxylin and eosin staining (Fig. 3).
Spleen sections from control infected mice showed infiltration of
lymphocytes and phagocytes, but bacteria were not observed. In contrast
to the spleen, the inflammatory response in the liver was more
localized, and only a slight increase in tissue damage was observed in
IFN--depleted mice compared with infected controls, suggesting that
the spleen is particularly sensitive to the necrotic effects of high
burdens of B. pseudomallei in vivo.
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Susceptibility of IFN--depleted mice to B. pseudomallei.
To identify the minimum infecting dose of B. pseudomallei, mice were challenged with serial 10-fold dilutions
of bacteria from 2 × 105 CFU or saline alone and
treated with either H22 or control L2 antibody. Mice given control MAb
showed no sign of illness and survived throughout the period of the
experiment with all bacterial inocula (Fig.
6). In contrast, all IFN--depleted
mice infected with 2 × 105 or 2 × 104 CFU died within 48 h. Furthermore, at the lower
inocula (2 × 103, 2 × 102, or
2 × 101 CFU), all anti-IFN--treated mice were dead
within 5 days of challenge. Even at an inoculum of 2 × 100 CFU, two of three anti-IFN--treated mice died,
whereas controls for this inoculum all survived. Finally, all
IFN--depleted mice given a further 10-fold dilution (i.e., 2 × 10
1 CFU) survived excluding any nonspecific
immunosuppressive effects of IFN- neutralization per se. The
accuracy of the infecting doses was confirmed by direct plating of the
bacterial suspension used in the challenge. Thus, under these
conditions, neutralization of IFN- lowered the effective lethal dose
of B. pseudomallei by 100,000-fold to approximately two
bacteria per animal. The importance of IFN- was confirmed in
experiments examining the sensitivity of IFN- receptor knockout mice
(G129) to infection with B. pseudomallei. Control wild-type
mice (129SVEV) were extremely susceptible to B. pseudomallei
infection, and at doses of from 105 to 102
CFU/mouse, both IFN-R+/+ and IFN-R/
mice all succumbed within 2 days. Nevertheless, when given
101 CFU per animal, two of three IFN-R+/+
mice died at day 12, whereas all IFN-R/ mice
succumbed within 2 days. Thus, by using either neutralizing MAb in
immunocompetent mice or animals lacking the IFN- receptor, functional loss of IFN- is associated with increased susceptibility to infection with B. pseudomallei.
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) CFU/mouse. Injection of the
antibodies into uninfected mice (
) was performed as a negative
control. Solid symbols represent mice treated with H22 MAb, and open
symbols represent L2 treatment. Mice were observed daily for 14 days
and the percent survival was plotted against time. Where multiple
experimental groups all showed 100% survival, data points have been
separated for clarity of presentation. All infected mice treated with
2 × 100 CFU (P < 0.05) and higher
doses of B. pseudomallei (P < 0.02)
resulted in a significantly earlier time to death when compared to
B. pseudomallei plus L2 control antibody. The results are
representative of two independent experiments.
The role of TNF- and IL-12 in resistance to acute infection with
B. pseudomallei.
In other model systems, the rapid
production of IFN- is initiated by the production of TNF- and
IL-12 (32). In order to assess the role of these cytokines
in melioidosis, TO mice were infected with B. pseudomallei
as previously described and given neutralizing MAb specific for either
TNF (TN3.19.12), IL-12 (C17.8), or IFN- at the time of infection. As
expected, neutralization of IFN- again resulted in 100% mortality
within 48 h (Fig. 7). However,
depletion of IL-12 resulted in acute lethal sepsis in four of five
animals by 3 days, and three of five animals died by 4 days after
neutralization of TNF in vivo. These results suggest that IL-12 and
TNF-, as well as IFN-, are also important mediators of resistance
to acute infection.
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) at a dose of 300 µg/mouse at the time of i.p. infection with
2 × 105 CFU of B. pseudomallei. All
infected mice treated with either H22 (P < 0.002),
anti-TNF (P < 0.005), or anti-IL-12 MAb (P < 0.005) resulted in a significantly earlier time to death
compared to B. pseudomallei plus control antibodies. Results
are presented as the percent survival against time. Where multiple
groups all showed 100% survival, data points have been separated for
clarity of presentation. The results are representative of two
independent experiments.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this report, we have generated murine models of both acute and
chronic infection to investigate the mechanisms of protective immunity
against the causative agent of human melioidosis. Three major findings
arose from this study. First, immunocompetent mice initially clear
viable B. pseudomallei from infected tissues but then enter
a chronic phase of infection which is invariably lethal after 2 to 16 months. Second, this early control mechanism is absolutely dependent
upon the rapid production of IFN-. Finally, TNF and IL-12 also play
a role in acute resistance to B. pseudomallei in vivo.
Our initial experiments demonstrated that inbred BALB/c mice are relatively susceptible to acute infection with B. pseudomallei, whereas the outbred TO strain was approximately 100-fold more resistant. Furthermore, 129SVEV mice showed even greater susceptibility, with a 50% lethal dose of ca. 100 CFU. Genetic differences in resistance have also been reported in other gram-negative bacterial infections (14), although the basis of the different susceptibilities of the animals to B. pseudomallei has yet to be clarified. The extreme susceptibility of some species to experimental infection with B. pseudomallei has previously hampered investigation of the protective immune response to this organism. Our ability to challenge TO mice with significant numbers of bacteria and generate either an acutely lethal infection or a chronic infection provided us with a practicable model to dissect the involvement of macrophage-activating cytokines on resistance in vivo. A kinetic study of bacterial clearance from the target organs of sublethally infected TO mice showed little evidence of bacterial growth but did show a progressive decline in viable B. pseudomallei over time. Clearance was marginally slower in the liver compared to the spleen, but in both organs 99% of bacteria were removed within 4 days after infection, and the majority of animals progressed to apparent sterilization of these organs by 28 days after infection. This result was associated with progressive splenomegaly and the rapid development of granulomas containing multinucleated giant cells in infected tissues, a feature characteristic of both human melioidosis and infection in other animal species (9, 30, 36). These observations support our recent description of the ability of Burkholderia species (both B. pseudomallei and B. mallei) to induce cell fusion and giant cell formation in macrophage cell lines in vitro (14a).
Two additional features of infection with B. pseudomallei are worthy of mention. First, splenic abscesses containing a high number of bacteria were observed in some animals which were otherwise asymptomatic and had negligible numbers of bacteria in other organs, indicating that B. pseudomallei can replicate in a focal manner without causing systemic toxicity. Second, at the doses used in this study, all animals that survived the initial infection entered a chronic phase lasting from 2 to 16 months before ultimately progressing to reactivation and death. The cellular and organ location of the few remaining bacteria which go on to cause reactivating disease have not been identified. However, reactivating lesions were observed at multiple tissue sites, including the foot pad and subcutaneous tissue, as well as in the main target organs of liver and spleen. These features mimic the characteristics of clinical melioidosis, further supporting the relevance of the murine model to the human disease (8).
When mice were depleted of IFN- at the time of infection, resistance
to B. pseudomallei was completely abolished and all animals
died within 40 h, whereas the control infected group survived for
several months. Neutralization of IFN- blocked the generation of
multinucleated giant cells (data not shown), increased the bacterial
burden in the spleen and liver by 4,400- and 8,500-fold, respectively,
and caused massive destruction of splenic lymphoid architecture. Under
these conditions, large numbers of bacteria were observed both
extracellularly and intracellularly within macrophages but, unlike the
case of Listeria monocytogenes (27), there was no
evidence that B. pseudomallei invades and replicates in
hepatocytes. The fact that as few as two CFU were capable of causing
lethal melioidosis in the absence of IFN- implies that resident
phagocytic cells such as peritoneal macrophages and Küpffer cells
are unable to kill B. pseudomallei in vivo. This idea is supported by reports of intracellular survival of B. pseudomallei in both resident macrophages and PMNL in vitro
(11, 16, 25). As yet, we have not identified the mechanisms
by which IFN- mediates its protective effects. IFN- is an
established activator of macrophage microbicidal activity and mediates
resistance against a wide range of other intracellular pathogens,
including Listeria spp., Salmonella spp., and
Mycobacterium spp., as well as some protozoan parasites (17). Recently, Miyagi and colleagues have demonstrated that IFN- activates a murine macrophage cell line to kill B. pseudomallei in vitro by a combination of both oxidative and
NO-mediated mechanisms (22). The data presented here are
consistent with these in vitro findings and provide direct evidence for
the importance of the IFN- response in vivo. That IFN- depletion
increases the susceptibility of mice to B. pseudomallei by
more than 100,000-fold (in terms of 50% lethal dose) clearly makes
melioidosis one of the most sensitive models of IFN--dependent
resistance described to date.
The rapidity with which anti-IFN- MAb abolishes resistance suggests
an innate rather than an acquired source of this cytokine in the
initial phase of infection. H22 MAb-treated mice showed signs of
illness within 18 to 24 h, suggesting that even in a naive host,
B. pseudomallei stimulates IFN- production within this
period. To date, there is no information on T-cell responses to this
organism in either humans or experimental animals. However, such rapid
IFN- responses are unlikely to be mediated by the clonal expansion
of antigen-specific T cells. Natural killer (NK) cells are an
alternative source of IFN- and represent the first secreting cells
after infection with other intracellular pathogens (2, 10,
28). Several pieces of evidence suggest this pathway may
contribute to resistance against B. pseudomallei. First,
other gram-negative bacteria, such as Salmonella spp. and
Francisella spp., can also stimulate T-cell-independent
IFN- responses (12, 26). Second, as shown here,
neutralization of either TNF- or IL-12, which stimulate secretion of
IFN- by NK cells, increases susceptibility to B. pseudomallei. Third, SCID mice infected with B. pseudomallei show an intermediate pattern of resistance between that of infected versus IFN--depleted mice, surviving up to 20 days
(data not shown). This pattern of partial resistance in SCID mice is
typical of other pathogens which are known to activate the NK cell
IFN- pathway (2). Thus, T cells are not required for
initial protection against lethal melioidosis but are absolutely necessary for maintenance of the chronic phase of infection. Finally, irradiated B. pseudomallei activate naive SCID spleen cells
(which are enriched in NK cells but devoid of both /
and /
T cells) for secretion of IFN- in vitro (19a). Together,
these results point to an initial, T-cell-independent pathway of
resistance prior to the generation of antigen-specific immunity. It
should be noted that TNF- and IL-12 have other important
proinflammatory actions, including the upregulation of adhesion
molecules and the promotion of Th1-type T-cell responses, which may
also contribute to resistance in the immunocompetent host
(17). Finally, the course of chronic infection may also
be influenced by the production of IFN- since in preliminary
experiments neutralization of IFN- promoted reactivation and
shortened the duration of the latent state (data not shown).
Experiments are in progress to identify the cellular source of IFN-
in the chronically infected host, which is likely to be derived from
antigen-specific T cells rather than NK cells (31).
In conclusion, the results presented here provide the first evidence
for the cellular basis of protective immunity against B. pseudomallei. A rapid IFN- response protects the naive host from acute sepsis after initial contact with the organisms and allows
progression into the chronic phase of the disease. This distinguishes
B. pseudomallei from immunity to L. monocytogenes, the classical paradigm of IFN--mediated
resistance, where infection leads to complete and protective
sterilizing immunity (33). Instead, melioidosis shares
characteristics with other pathogens, such as M. tuberculosis and Toxoplasma gondii, where a successful IFN- response is still unable to eliminate the organism, placing the
host at risk of reactivation if immune competence becomes impaired at a
later time. It will be interesting to determine whether sterilizing
immunity to B. pseudomallei can be achieved under other
circumstances, such as after cytokine therapy with exogenous IFN- or
IL-12 or after vaccination. To date, no information is yet available on
the nature of protective immunity in humans infected with this
organism. However, patients presenting with lethal melioidosis express
high levels of circulating cytokines, including TNF- and IFN-,
which correlate with mortality (3, 29). Animal models of
septic shock indicate that the same cytokines which confer resistance
when produced in a controlled and localized manner (including IFN-,
TNF-, and IL-12) promote pathology when produced systemically to an
overwhelming bacterial load (1, 4, 15). From the data
presented here, we predict that the generation of IFN--dominant NK
cell and T-cell responses will be essential for the control of acute
human melioidosis and entry into the chronic phase. Experiments to test
this hypothesis and assess the effect of known risk factors for
reactivation (such as diabetes) on these responses should provide a
better understanding of the immunological processes which control
infection with this important pathogen.
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ACKNOWLEDGMENTS |
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This work was supported by the Wellcome Trust (project grant 034656/2/91/2). P.S. is supported by grant from the Ministry of Sciences and Technologies of Thailand.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 44-171-927-2361. Fax: 44-171-637-4314. E-mail: g.bancroft{at}lshtm.ac.uk.
Editor: S. H. E. Kaufmann
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, July 1999, p. 3593-3600, Vol. 67, No. 7
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