Infection and Immunity, June 2000, p. 3748-3753, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Administration of Endotoxin Associated with Lipopolysaccharide
Tolerance Protects Mice against Fungal Infection
Naïma
Rayhane,1
Catherine
Fitting,1
Olivier
Lortholary,2
Francoise
Dromer,2 and
Jean-Marc
Cavaillon1,*
Unité
d'Immuno-Allergie1 and Unité de
Mycologie,2 Institut Pasteur, 75015 Paris,
France
Received 5 January 2000/Returned for modification 1 March
2000/Accepted 10 March 2000
 |
ABSTRACT |
Lipopolysaccharide (LPS) pretreatment of mice resulted in a
significantly enhanced survival after disseminated Cryptococcus neoformans infection. The survival was associated with reduced fungal burden in tissues. LPS-pretreated mice had lower levels of
cytokines in blood, spleen, and lungs and higher levels in brain.
Pentoxifylline abolished the beneficial effect of LPS pretreatment.
 |
TEXT |
Whether the induction of endotoxin
tolerance leads to an enhanced sensitivity to infection remains a
challenging question. The data reported in the literature are rather
controversial. Deleterious effects of endotoxin tolerance were
suggested by a study in which pretreatment of mice with
lipopolysaccharide (LPS) resulted in a lower titer of plasma interferon
after challenge with Newcastle disease virus (26). Although
this result may suggest a reduced ability of the animals to control
viral infections, this question was not addressed in that study.
Similarly, using a peritonitis model, it was reported that
monophosphoryl lipid A pretreatment attenuated the levels of
circulating cytokines and reduced the neutrophil margination in tissues
(22). The efficiency of this pretreatment, however, was not
addressed in terms of outcome. It was also reported that a few hours
after LPS exposure, pretreated mice had an impaired capacity to clear Pseudomonas aeruginosa from their lungs (13).
Injection of heat-killed gram-negative bacteria in baboons 12 h
before an intravenous infusion of viable bacteria led to increased lung
injury (25). In in vitro experiments it was observed that
preexposure of macrophages to LPS resulted in reduced leishmanicidal
activity (24). Furthermore, macrophages previously exposed
to LPS are less responsive to various types of stimuli, including
bacterial superantigens (16) and Staphylococcus
aureus, Streptococcus pyogenes, or zymosan
(2). In contrast, endotoxin tolerance was shown to be
beneficial in preventing mortality after thermal injury (7),
in protecting against hepatic and renal ischemia reperfusion (3,
8), and in reducing the size of myocardial infarction initiated
by coronary occlusion (5).
No studies have yet addressed the consequences of endotoxin tolerance
in terms of protection against fungal pathogens. Since disseminated
Cryptococcus neoformans infection occurs in severely immunocompromised patients (15) who are likely to encounter other microorganisms, it was of interest to analyze the influence of
endotoxin pretreatment on survival, fungal burden, and cytokine expression in blood and tissues after intravenous inoculation of
C. neoformans.
Six- to 7-week old male BALB/c mice were made LPS tolerant as
previously described (18) with intravenously injections of 2.5 µg of Escherichia coli LPS (0111:B4) (Sigma) in 0.1 ml
of saline daily for 2 days and were intravenously inoculated on the third day with C. neoformans (2 × 106 live
cells per mouse) (19). Control mice were injected with the
same volume of saline. Blood samples were obtained by cardiac puncture,
and lungs, spleens, and brains were excised. Determination of CFU,
cytokine measurements, and analysis of nitrite and nitrate in blood and
tissues were performed as previously described (19).
Pretreatment of mice with LPS was associated with a protective effect
against a subsequent fungal challenge in terms of survival and fungal
burden in blood and tissues (Fig. 1 and
2). The fungal burden was always higher
in control mice than in the LPS-tolerant animals in the late days of
the follow-up in all analyzed compartments and 1 day after infection in
the lungs, the brain, and the blood. In contrast, a significantly and
reproducibly higher number of CFU was observed on day 3 in the spleens
of tolerant animals.

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FIG. 1.
Survival curves for control and LPS-pretreated (2.5 µg
for 2 days) mice following intravenous infection with 2 × 106 C. neoformans (Cn) cells. The data,
expressed as percent surviving mice, are the cumulative results of four
different experiments including a total of 51 mice in each group.
Median survival times for control and LPS-tolerant mice were 22 and
>49 days, respectively. The significance of the Kaplan-Meier survival
curves was assessed by the Mantel-Cox log rank test (P < 0.0001).
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FIG. 2.
Kinetics of CFU counts in spleen, lungs, brain, and
blood after inoculation with C. neoformans (Cn) in normal
and LPS-pretreated mice. The results are expressed as the means and
standard errors of the means for seven mice in each group and are
representative of three independent experiments. Significance was
assessed by the Mann-Whitney U test. D, day.
|
|
A survey of cytokine expression was performed over 13 days (data not
shown). During the later course of infection, on day 13, lower levels
of tumor necrosis factor alpha (TNF-
), KC chemokine, gamma
interferon (IFN-
), interleukin-10 (IL-10), and nitrite and nitrate
in the blood; of TNF-
, IL-1
, IL-6, and IFN-
in the spleen; and
of TNF-
, IL-1
, and IL-10 in the lungs were observed in
LPS-tolerant animals (Fig. 3). This
observation most probably reflects the lower fungal burden in these
tissues in tolerant mice. Interestingly, a reversed cytokine pattern
was observed in the brains of tolerant mice, with higher levels of
TNF-
, KC, IL-10, and IL-12. These results further illustrate that
the brain behaves differently in terms of its cytokine network than the peripheral tissues (11) and that higher expression of
certain cytokines in the brain was associated with protection
(19).

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FIG. 3.
Levels of IL-1 , KC, TNF- , IL-6, IFN- , IL-10,
IL-12, and nitrite and nitrate (NOx) in different compartments on day
13 postinfection in normal and LPS-pretreated mice. Cn, C. neoformans. The results are expressed as the means and standard
errors of the means for 7 to 13 individual mice in each group. *,
P < 0.05; **, P < 0.01;
***, P < 0.001.
|
|
An early induction of cytokines, particularly TNF-
, during the LPS
pretreatment may explain the observed protection. This was further
suggested by experiments using pentoxifylline, an inhibitor of
phosphodiesterases, which is known to prevent TNF production (6,
23). Pentoxifylline diminished the protective effect of the LPS
pretreatment and by itself accelerated the occurrence of death in
C. neoformans-infected mice, confirming the protective role
of TNF in this fungal infection (1, 4, 9, 10, 19) (Fig.
4). To assess the role of IL-1, IL-1
receptor antagonist (200 µg/mouse) was injected in another group of
animals 1 h before the LPS pretreatment. In our model IL-1ra
failed to reverse the protective effect of the injection of endotoxin
against C. neoformans infection (data not shown).

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FIG. 4.
Survival curves for normal, LPS-pretreated,
pentoxifylline (PXF)-pretreated, and pentoxifylline-plus LPS-pretreated
mice after C. neoformans (Cn) fungal infection. The data are
the cumulative results of two independent experiments including a total
of 20 mice. PXF was injected 30 min before LPS or fungi. Control
experiments revealed 8,151 ± 1,384 pg of TNF per ml in blood 90 min after LPS injection (100 µg/mouse), whereas only 707 ± 129 pg/ml was found in PXF-pretreated mice. For LPS-C.
neoformans versus PXF-LPS-C. neoformans, P < 0.0001; for LPS-C. neoformans versus PXF-C.
neoformans, P < 0.0001. For PXF-C.
neoformans versus saline-C. neoformans, the median
survival times were 17 and 23 days, respectively, and P = 0.05 when the comparison was performed between days 13 and 25.
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|
We also investigated whether delayed LPS injection could be beneficial.
Thus, mice were injected 1, 3, and 8 days after the C. neoformans infection. As shown in Fig.
5A, no significant alteration of survival
curves was observed when 2.5 µg of LPS was employed. In contrast, a
protective effect was induced when a larger amount of LPS (25 µg) was
injected 1 day after the fungal challenge (Fig. 5B). No protective
effect was seen when the injection was performed 3 days later, and the
injection even proved to be lethal when performed 8 days after
infection, reminiscent of the increased toxicity of LPS when delivered
with other microbial agents (17) or even with TNF
(21).

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FIG. 5.
Survival curves for mice infected with C. neoformans (Cn) and receiving either no LPS or 2.5 µg (A) or 25 µg (B) of LPS on day (D) 1, 3, or 8. The data are the results of
experiments performed with 10 mice in each group. In panel B, for day 1 versus control P = 0.01; for day 8 versus control,
P < 0.0001.
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|
Our observations suggest that LPS pretreatment, which is known to be
associated with the induction of a state of anergy, is also responsible
for a priming effect after the production of cytokines that are known
to be protective upon a further microbial challenge. Indeed, many
authors have demonstrated that injection of IL-1 or TNF was protective
against further microbial challenge, including fungal infection
(10, 14). These protective effects may reflect the
activation of microbicidal activity by mononuclear phagocytic cells
(4, 20). It has also been shown that polymorphonuclear neutrophils (PMN) may be primed following LPS pretreatment
(12). Since it has been reported that PMN are active in
killing of C. neoformans, one can hypothesize that part of
the efficient resistance to C. neoformans conferred by LPS
pretreatment may reflect an enhanced microbicidal activity of PMN.
Interestingly, the beneficial effect of LPS was associated with
delivery either before or soon after the inoculation, further
supporting the concept of a priming effect.
Although this model suggests that pretreatment with LPS may be of some
advantage in preventing the development of an infection, one should be
very careful before extrapolating these results for prophylactic
purposes. Thus, on day 3 after inoculation, the pretreated animals had
a reproducible higher CFU count in the spleen that was associated with
a higher level of TNF, and late injection of LPS resulted in a shorter
survival. In a model of viral infection with lymphocytic
choriomeningitis virus, we failed to demonstrate any beneficial effect
of LPS pretreatment (unpublished observation). Together our results
suggest that LPS exposure can induce both a hyporeactive state,
illustrated by the endotoxin tolerance phenomenon, and a priming effect
which may favor antiinfectious responses.
 |
ACKNOWLEDGMENTS |
O.L. was a recipient of a fellowship from SIDACTION and Assistance
Publique-Hôpitaux de Paris. Part of the work was supported by a
grant "Contrat Interne de Recherche Clinique" from the Pasteur Institute to Françoise Dromer.
We thank Jacques Callebert (Service de Biochimie, Hôpital
Lariboisière) for the NOx measurements. We express our gratitude to Ernst Rietschel for his helpful comments and for reviewing the manuscript.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité
d'Immuno-Allergie, Institut Pasteur, 28 rue Dr. Roux, 75015 Paris,
France. Phone: 33 1 45 68 82 38. Fax: 33 1 40 61 31 60. E-mail:
jmcavail{at}pasteur.fr.
Editor:
T. R. Kozel
 |
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Infection and Immunity, June 2000, p. 3748-3753, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.