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International Immunology Advance Access originally published online on October 3, 2007
International Immunology 2007 19(11):1303-1311; doi:10.1093/intimm/dxm101
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Divergent roles of IFNs in the sensitization to endotoxin shock by lactate dehydrogenase-elevating virus

Thao Le-Thi-Phuong1, Laure Dumoutier1,2, Jean-Christophe Renauld1,2, Jacques Van Snick1,2 and Jean-Paul Coutelier1

1 Unit of Experimental Medicine
2 Ludwig Institute for Cancer Research, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, UCL MEXP 7430, Avenue Hippocrate 74, 1200 Bruxelles, Belgium

Correspondence to: J.-P. Coutelier; E-mail: jean-paul.coutelier{at}uclouvain.be


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
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The effect of mouse infection with lactate dehydrogenase-elevating virus (LDV), a usually non-pathogenic virus, on concomitant bacterial endotoxin shock was analyzed, in terms of lethality and cytokine production. A strong enhancement of susceptibility to the shock was observed in mice acutely infected with this virus. It correlated with a sharp increase of tumor necrosis factor and leukemia inhibitory factor production and was controlled by the mouse genetic background. The viral infection led to an imbalance in the cytokine response to LPS, with an enhancement of pro-inflammatory cytokines, including IL-18 and IFN-{gamma} and a delayed secretion of anti-inflammatory IL-10 that could result in exacerbated macrophage activation. Enhanced IFN-{gamma} production was involved in the virus-induced susceptibility to shock. In sharp contrast with other viral infections, IFN-{alpha}/ß diminished IFN-{gamma} production and the resulting increased response to LPS in LDV-infected animals.

Keywords: cytokines, LPS, macrophages


   Introduction
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 Abstract
 Introduction
Methods
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Both genetic and environmental factors have been shown to determine the outcome of sepsis and septic shock. Among these factors, co-infection with viruses such as influenza or respiratory syncytial virus largely increases both the frequency and the severity of bacterial infections (1). Experimental models have shown an enhanced susceptibility of mice to concomitant exposure to LPS and infection with agents including lymphocytic choriomeningitis virus (LCMV), adenovirus, Theiler's virus or vesicular stomatitis virus (2–6). Type I, and to a lesser extent type II IFNs have been reported as major mediators of this exacerbation of endotoxin shock by viruses (2–5). However, since these viruses are pathogenic per se, or may at least induce pathology when appropriate circumstances are met, it is difficult to discriminate between an exacerbation of LPS-induced pathogenicity by the viral agents and between the enhancement or induction of viral pathology through mechanisms triggered by the endotoxin.

Lactate dehydrogenase-elevating virus (LDV) is an arterivirus, non-pathogenic per se in most strains of infected mice because, in these animals, its tropism is restricted to a sub-population of non-vital macrophages. However, LDV triggers a rapid but transient burst of cytokine production that may interfere with ongoing pathologic mechanisms initially unrelated to the infection [reviewed in (7)]. It provides, therefore, a unique model to analyze the consequences of a concomitant viral infection on cytokine-mediated diseases like endotoxin shock. Here, we report that LDV, like other viruses, dramatically increases the susceptibility of infected hosts to LPS shock. This sensitization that is controlled by genetic factors is correlated with unbalanced production of pro-inflammatory cytokines and anti-inflammatory IL-10. This virally induced exacerbation of endotoxin shock is partially mediated by IFN-{gamma}, but is strongly enhanced in animals unresponsive to type I IFNs.


   Methods
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 Abstract
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 Methods
Results
 Discussion
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 References
 
Animals
129/Sv, BALB/C and DBA/2 female mice were bred at the Ludwig Institute for Cancer Research (Brussels) by G. Warnier and used when 8–12 weeks old. IFN-{gamma} receptor-deficient mice (G129), received by courtesy of F. Brombacher (Max Planck Institute for Immunobiology, Freiburg, Germany) were reared in the same manner as the 129/Sv animals, from which they were initially derived by S. Huang and M. Aguet (8). IFN-{alpha}/ßR0/0 mice were also originally derived by M. Aguet (9). The project was approved by the local commission for animal care.

Virus
Mice were infected by intra-peritoneal (i.p.) injection of ~2 x 107 50% infectious doses (ID50) of LDV (Riley strain; American Type Culture Collection, Rockville, MD, USA) in 500 µl saline.

LPS
LPS from Escherichia coli (E. coli 0111:B4; Sigma, Bornem, Belgium) was injected i.p. in 500 µl saline. Sensitivity to endotoxin shock was determined by injecting groups of mice with titrated doses of LPS. LD50 were calculated following the method of Reed and Muench (10).

Cytokine assays
Tumor necrosis factor (TNF) was measured by bioassay as described previously (11, 12). Briefly, WEHI-164 clone 13 cells were incubated in the presence of actinomycin D with samples to be tested in medium supplemented with LiCl to maximize sensitivity to TNF-mediated cytotoxicity. Cell viability was then assessed with a tetrazolium-based colorimetric assay and TNF concentrations were calculated in reference to the maximal cell lethality obtained with recombinant cytokine (kind gift of W. Fiers, University of Gent, Belgium). 1 U ml–1 was arbitrarily defined as the TNF concentration that induced 50% cell lethality (corresponding to ±1 pg ml–1).

IFN-{gamma} was measured by ELISA using a commercial kit (CytoSets®), following the manufacturer's recommendations (Biosource, Camarillo, CA, USA). IL-12 and leukemia inhibitory factor (LIF) were assayed with Quantikine® kits (R&D Systems, Minneapolis, MN, USA). IL-1ß and IL-10 were measured using capture and detection antibodies and standards from DuoSet® kits (R&D Systems) with streptavidin–HRP from Amersham (Little Chalfont, UK) and Ultra-TMB-ELISA® from Pierce (Rockford, IL, USA). An ELISA kit from MBL (Nagoya, Japan) was used to measure IL-18 levels.

Plasma IL-22 concentrations were measured by ELISA using a murine mAb directed against human IL-22 (MH22B2) and an affinity-purified polyclonal rabbit antibody against murine IL-22. MH22B2 cross-reacts with murine IL-22. Briefly, microtiter plates (Immunoplates, Nunc, Roskilde, Denmark) were coated with MH22B2 (5 µg ml–1) in 20 mM glycine, 30 mM NaCl (pH 9.2) buffer and incubated overnight at 4°C. The plates were then washed in PBS with Tween 20 (5 x 10–4) and blocked in PBS–BSA (5%) for 1 h at 37°C. After washing, sample dilutions in PBS with 2.5% BSA were added to the plates, which were incubated for 3 h at 37°C. A biotinylated rabbit polyclonal anti-mIL-22 antibody (0.136 µg ml–1) was used as secondary antibody for a 2 h incubation at 37°C. Plates were then washed and soaked for 7 min in NaCl containing 1% NP-40 (Fluka AG, Buchs, Switzerland). Binding of the second antibody was detected using streptavidin–HRP (Amersham). The assay was developed by adding 50 µl of Ultra-TMB-ELISA® (Pierce) and the reaction stopped with 50 µl of 2 M H2SO4 before reading the absorbance at 450 nm.

IL-6 was assayed by incubation of serial sample dilutions with the mouse IL-6-dependent B cell hybridoma 7TD1 (2000 cells per microwell) in 0.2 ml Iscove’s medium containing 10% FCS and supplemented with 0.24 mM L-asparagine, 0.55 mM L-arginine, 1.5 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 0.1 mM hypoxanthine and 0.016 mM thymidine. Viability was assessed 4 days later by hexosaminidase determination. Results, expressed in U ml–1, were defined as the concentration producing half-maximal growth of the cells (13).

Antibody
Goat anti-TNF antiserum was prepared in the Hormonology Laboratory of the Centre d'Economie Rurale (Marloie, Belgium) after a first immunization with 20 µg recombinant mouse TNF, followed by monthly immunizations with 10 µg TNF. Polyclonal antibody was prepared by precipitation with ammonium sulfate.

Flow cytometry
Macrophage populations were analyzed by flow cytometry, as described (14) with a fluoresceinated anti-mouse F4/80 mAb (Serotec, Raleigh, NC, USA).

Statistical analysis
When appropriate, statistical analysis was performed using non-parametric unpaired two-tailed Mann–Whitney test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Exacerbation of endotoxin pathogenicity by LDV infection
Survival of either uninfected 129/Sv mice or animals acutely infected with LDV was monitored after administration of titrated doses of LPS. As shown in Fig. 1, uninfected mice were rather resistant to endotoxin shock. In contrast, LDV-infected animals displayed a strikingly enhanced susceptibility to LPS. This enhanced susceptibility to endotoxin shock was observed in mice infected for 1 and 2 days with LDV, but not as much in animals infected for 4 or 7 days (data not shown).


Figure 1
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  		Fig. 1. Enhancement by LDV infection of mouse susceptibility to endotoxin shock. Survival was monitored after injection of titrated doses of LPS to groups of six 129/Sv mice, 24 h after administration of either saline or LDV.

 
Since TNF plays a major role in the development of endotoxin shock, the consequences of LDV infection on its production in response to LPS administration were analyzed. Our results showed a synergistic effect of LPS and LDV on the secretion of this cytokine (Fig. 2A). LDV infection led to very low TNF titers, with plasma levels below 100 U ml–1 in all tested mice, at 1 day after infection. At 2 h after administration of 24 µg LPS, uninfected mice developed a modest TNF response. In contrast, TNF levels were dramatically enhanced in LDV-infected animals that received the same dose of LPS. TNF levels then quickly decreased to reach much lower values at 6 h after LPS administration (Table 1).


Figure 2
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  		Fig. 2. Synergistic effect of LDV infection and LPS administration on TNF production. (A) TNF was measured in plasma of 129/Sv mice 2 h after administration of 24 µg LPS or saline. LDV infection was performed 24 h before LPS administration. Results are shown for groups of three to four mice (mean ± SEM). In this experiment, the limit of detection was 6 U ml–1. (B) In vitro TNF production by peritoneal cells. Peritoneal cells freshly obtained from either uninfected 129/Sv mice or animals infected with LDV for 1 day, without other in vivo stimulation, were incubated for 4 h with medium alone (hatched columns) or with LPS (10 ng ml–1, closed columns). TNF was assayed in individual supernatants (results shown as mean ± SEM for groups of four to five animals).

 

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  		Table 1. Kinetics of cytokine production in LDV-infected mice in response to LPS

 
To determine whether LDV could sensitize cells to TNF production, we analyzed the effect of LDV infection on subsequent in vitro secretion of this cytokine by peritoneal cells in response to LPS. Flow cytometry analysis of peritoneal cells showed that LDV infection did not increase the macrophage proportion in these cells (5.9 ± 2.6% in infected animals versus 9.7 ± 1.8% in control mice, P = 0.3429). TNF secretion was significantly enhanced when peritoneal cells derived from LDV-infected mice were incubated with LPS, as compared with that of similar cells obtained from control uninfected mice (Fig. 2B, P = 0.0317). However, this increase of in vitro TNF production by peritoneal cells from LDV-infected mice was not as dramatic as the synergistic enhancement of this cytokine production observed in vivo after administration of both LPS and LDV.

Since the peak of TNF production occurred in LDV-infected mice 2 h after LPS administration, we measured the effect of the virus on the secretion of other cytokines at the same time. IL-1ß production was induced by LPS, but not LDV administration alone (Fig. 3A). No significant difference in the early IL-1ß response to LPS was observed between uninfected and infected animals (P = 0.3524). Similarly, LDV infection did not increase significantly (P = 0.3) the IL-22 secretion that was induced 2 h after LPS injection (Fig. 3C). Although it may be secreted at earlier times after LDV infection, little, if any, IL-6 production was found at 1 day after viral inoculation. However, production of this cytokine was quickly triggered by LPS administration at higher levels in LDV-infected mice than in control animals (Fig. 3B, P = 0.0022). An early dramatic enhancement of LIF secretion in response to LPS injection was also observed in LDV-infected animals, when compared with control mice (Fig. 3D).


Figure 3
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  		Fig. 3. IL-1ß, IL-6, IL-22 and LIF production after LPS and LDV administration to 129/Sv mice. IL-1ß, IL-6, IL-22 and LIF were measured in plasma of 129/Sv mice at 2 h after administration of 30 µg (IL-1ß, LIF) or 100 µg (IL-6, IL-22) LPS (independent experiments). LDV infection was performed 24 h before LPS administration. Results are shown for groups of four to six mice (mean ± SEM). Limits of detection are indicated.

 
Whereas IFN-{gamma} levels were below the limit of detection in plasma of control mice, irrespective of the administration of LPS, the production of this cytokine, which was induced by the infection alone, was strongly enhanced in LDV-infected mice that also received LPS (Fig. 4A, P = 0.0381). At 1 day after inoculation, LDV triggered only little IL-12 production. The virus did not enhance the secretion of this cytokine that was induced at 2 h after LPS administration (Fig. 4B, P = 0.6095). Contrasting with IL-12, IL-18 plasma levels were increased by LDV (Fig. 4C) with a peak at 1 day after infection (data not shown). A modest IL-18 secretion was also induced by administration of LPS to uninfected mice. This IL-18 production was markedly increased in LDV-infected animals (Fig. 4C, P = 0.0095).


Figure 4
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  		Fig. 4. IFN-{gamma}, IL-12, IL-18 and IL-10 production after LPS and LDV administration to 129/Sv mice. IFN-{gamma}, IL-12 and IL-18 were measured in plasma of 129/Sv mice at 2 h after administration of 100 µg LPS. LDV infection was performed 24 h before LPS administration. Results are shown for groups of four to six mice (mean ± SEM). Limits of detection are indicated. IL-10 was measured in an independent experiment in plasma of 129/Sv mice at different times after administration of 10 µg LPS. LDV infection was performed 24 h before LPS administration. Results are shown for groups of four to five mice (mean ± SEM).

 
Contrasting with the production of pro-inflammatory cytokines, IL-10 secretion was either significantly decreased or unchanged in LDV-infected 129/Sv mice, at least at an early time (2 h) after LPS administration (Fig. 4D, P = 0.0317). However, at later times after LPS injection, IL-10 production decreased in uninfected animals whereas it increased in infected mice and reached levels significantly higher than in controls (Fig. 4D, P = 0.0159). IL-10 could not be detected in the plasma of neither control nor infected mice that did not receive LPS administration (data not shown).

Finally, no effect of LDV infection on transforming growth factor-ß1, IL-1{alpha}, IL-4, IL-9, IL-13 and IL-17 production was detected (data not shown).

In addition to the delay in IL-10 secretion pattern and to the dramatic drop in TNF production, a few changes were observed in the later cytokine response to LPS in LDV-infected mice. Between 2 and 6 h after LPS administration, a significant enhancement in IL-1ß and LIF production, and to a lesser extent in IL-6, was observed in LDV-infected animals while IL-10 levels kept increasing. In contrast, no significant differences in IFN-{gamma}, IL-12 and IL-18 secretion were found (Table 1).

At 4 h after LPS injection, TNF production in LDV-infected mice correlated with the dose of LPS that was injected (Table 2). In contrast, a plateau was quickly reached for IFN-{gamma}, IL-1ß, LIF, IL-10 and IL-18 production.


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  		Table 2. Cytokine production in LDV-infected mice in response to different LPS doses

 
Role of TNF and IFN-{gamma} in LDV-enhanced endotoxin shock
To assess the role of TNF in the development of endotoxin shock enhanced by LDV, infected mice were treated with 5 mg anti-TNF goat IgG 1 h before administration of 33 µg LPS. While four of four control animals that received saline as treatment died after LPS injection, all four mice that received anti-TNF antibody survived. Anti-TNF treatment resulted also in a near complete suppression of LIF production 6 h after LPS injection, as shown in a distinct experiment (Fig. 5A, P = 0.0079). In contrast, the inhibition of IFN-{gamma} production in response to LPS in LDV-infected mice by the same anti-TNF treatment, although detectable, was not quite significant (Fig. 5B, P = 0.0556).


Figure 5
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  		Fig. 5. Effect of anti-TNF treatment on cytokine production. LIF and IFN-{gamma} were measured in plasma of groups of 129/Sv mice at 6 h after administration of 30 µg LPS. LDV infection was performed 24 h before LPS administration. Mice received either saline or anti-TNF treatment 1 h before LPS administration. Results are shown for groups of five mice (mean ± SEM).

 
This enhancing effect of LDV infection on the susceptibility to LPS-induced shock was examined in different mouse strains (Table 3). High sensitivity to endotoxin shock after LDV infection was found in all mouse strains tested. The increase in susceptibility of LDV-infected mice, when compared with uninfected animals was greater in 129/Sv (52-fold reduction of a LD50) than in BALB/c or DBA/2 (7- and 5-fold, respectively). However, measurement of susceptibility to LPS in a large number of experiments indicated that LDV-infected DBA/2 mice were statistically less sensitive to LPS with a LD50 of 38 µg than infected 129/Sv and BALB/C animals for which LD50 were 11 and 18 µg, respectively (significant differences, P = 0.0001 and 0.0130, respectively). In contrast, sensitivity of uninfected 129/Sv, BALB/C and DBA/2 mice to LPS was rather low, with LPS LD50 above 100 µg.


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  		Table 3. Enhancement of mouse susceptibility to endotoxin shock by LDV infection in different mouse strains

 
The role of TNF in the susceptibility of LDV-infected mice to LPS was further suggested by the observation that the relative resistance of LDV-infected DBA/2 mice to endotoxin shock was reproducibly correlated with a decrease in plasma levels of TNF at early times after LPS injection, when compared with BALB/C animals (Fig. 6). At later times, TNF levels were similar in these two mouse strains.


Figure 6
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  		Fig. 6. TNF production after LPS administration to LDV-infected BALB/C and DBA/2 mice. TNF was measured in plasma of BALB/C or DBA/2 mice at different times after administration of 30 µg LPS. LDV infection was performed 24 h before LPS administration. Results are shown for groups of five mice (mean ± SEM). **Difference between BALB/c and DBA/2 mice, P = 0.0286; *not quite significant difference between BALB/c and DBA/2 mice, P = 0.0556.

 
IFN-{gamma} has been shown to be at least partly responsible for the enhancement of susceptibility to bacterial endotoxin shock in mice infected with other viruses (2, 3). To determine whether this cytokine played a similar role in LDV-enhanced sensitivity to endotoxin shock, we compared the effect of LPS administration in infected mice deficient for the receptor of this cytokine to their normal counterparts. The animals deficient for IFN-{gamma} receptor showed an increased resistance to LDV-enhanced endotoxin shock (Fig. 7).


Figure 7
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  		Fig. 7. Resistance of IFN-{gamma} receptor-deficient mice to LDV-enhanced endotoxin shock. Survival was monitored after injection of titrated doses of LPS to groups of four 129/Sv (open symbols) mice or IFN-{gamma} receptor-deficient mice (G129, closed symbols), 24 h after administration of LDV.

 
Role of IFN-{alpha}/ß in LDV-enhanced susceptibility to endotoxin shock
Type I IFNs that are produced in the course of viral infections have been shown to play a major role in the sensitization by other viruses to LPS-induced shock (4, 5). To determine if they were similarly involved in LDV-induced increase in susceptibility to endotoxin, mice deficient in type I IFN receptor were infected with LDV, and then submitted to LPS administration. Surprisingly, these animals showed a mean susceptibility to the shock that was 4-fold higher than that of their normal counterparts (Fig. 8A, P = 0.0030). In contrast, no clear difference in susceptibility was found between uninfected 129/Sv and IFN-{alpha}/ßR0/0 mice (Fig. 8A).


Figure 8
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  		Fig. 8. Role of IFN-{alpha}/ß in the enhanced susceptibility of LDV-infected mice to LPS shock. (A) Survival was monitored after injection of titrated doses of LPS to groups of three to four 129/Sv and IFN-{alpha}/ßR0/0 mice, 24 h after administration of LDV. LPS LD50 are shown as mean ± SEM calculated from eight independent measurements for infected mice (different from the data set shown in Fig. 1), each of the experiments carried out with 129/Sv and IFN-{alpha}/ßR0/0 side by side. Results for uninfected mice were calculated from cumulative data of three independent experiments. (B) TNF was measured in plasma of 129/Sv (open symbols) and IFN-{alpha}/ßR0/0 mice (closed symbols) at different times after administration of 10 µg LPS. LDV infection was performed 24 h before LPS administration. Results are shown for groups of six mice (mean ± SEM). (C) IFN-{gamma} was measured in plasma of 129/Sv (hatched columns) and IFN-{alpha}/ßR0/0 mice (closed columns) at 2 and 6 h after administration of 10 µg LPS. LDV infection was performed 24 h before LPS administration. Results are shown for groups of six mice [same as (B), mean ± SEM]. (D) IL-12 and IL-18 were measured in plasma of 129/Sv (hatched columns) and IFN-{alpha}/ßR0/0 mice (closed columns) at 2 h after administration of 10 µg LPS. LDV infection was performed 24 h before LPS administration. Results are shown for groups of six mice [same as (B), mean ± SEM].

 
The enhanced susceptibility of LDV-infected IFN-{alpha}/ßR0/0 mice to LPS was correlated by a strong increase in TNF production with similar kinetics (Fig. 8B, P = 0.0022 at 2 h after LPS administration). As in wild-type animals, anti-TNF antibody treatment nearly completely prevented the development of lethal endotoxin shock in LDV-sensitized IFN-{alpha}/ßR0/0 mice (data not shown). IFN-{gamma} production was also significantly enhanced in IFN-{alpha}/ßR0/0 animals (P = 0.0022 and 0.0043 at 2 and 6 h after LPS administration, respectively), with a production of this cytokine that strongly increased with time (Fig. 8C). Interestingly, while IL-18 secretion was only marginally increased in IFN-{alpha}/ßR0/0 animals (Fig. 8D, P = 0.0649), a dramatic early enhancement of IL-12 was observed in these mice, when compared with 129/Sv counterparts (P = 0.0022).


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
We report here that, like other viruses (2–6), LDV dramatically sensitizes mice to LPS-triggered shock. Unlike these other viruses which, with their broader tropism, may have the potentiality to induce pathology under appropriate conditions, LDV is benign by itself in the mouse strains used here. This is due to its strictly restricted tropism for a sub-population of non-vital macrophages. It is therefore possible to ascribe the enhanced lethality observed in infected mice to a genuine exacerbation of LPS pathogenicity rather than to a mixed effect of combined endotoxin and virally induced lesions. Therefore, LDV provides a useful model to understand how a virus may modulate the sensitivity of its host to endotoxin shock.

This exacerbated susceptibility of LDV-infected mice to endotoxin was mostly mediated by an increased production of TNF, probably by activated macrophages. The role of TNF was shown by the enhanced in vitro response of peritoneal cells to LPS, by the in vivo correlation between LPS doses and TNF levels and especially by protection of infected animals with anti-TNF antibody. Moreover, this enhanced susceptibility of mice to endotoxin shock induced by the viral infection was at least partly controlled by genetic factors since some mouse strains, including BALB/C, were more sensitive than others, such as DBA/2 mice. This difference in susceptibility of infected mice, which was not observed in the uninfected corresponding animals as reported previously (15), was correlated with the production of TNF in response to LPS administration. Although other factors may also be involved, this result may fit with observations in patients that showed an association between polymorphism in the TNF-{alpha} promoter region, cytokine secretion in response to bacterial infection and outcome of septic shock (16).

So far, little was known on the production by virally infected mice exposed to endotoxin shock of early pro-inflammatory cytokines such as LIF, IL-1ß, IL-6 and IL-22 that are produced mostly by macrophages and T lymphocytes and which may contribute to the pathogenesis of the shock. The most conspicuous synergistic effect of LDV and LPS was on the production of LIF. However, the peak of LIF production was delayed in comparison to TNF kinetics and was suppressed by an anti-TNF antibody treatment, indicating that LIF may reflect increasing tissue damages resulting from the LDV-exacerbated disease (17), rather than primarily contribute to the pathogenesis of the shock by itself, as suggested in other models (18). Moreover, the consequences of LDV infection on LPS-induced production of IL-6, the role of which, in the pathogenesis of the shock, is disputed (19–21), of IL-22, a pro-inflammatory cytokine produced by Th17 cells, but that shares functional characteristics with IL-6 (22), and of IL-1 that may enhance TNF pathogenesis (23), were either less important or absent. This indicated that secretion of these cytokines may probably not explain the enhanced lethality of LPS in LDV-infected animals. Since macrophages are a major source of TNF, LIF, IL-6 and IL-1ß, it is interesting to see that the production of these cytokines in response to LPS administration is differentially regulated by LDV infection, which may correspond either to the involvement of diverse cell sub-populations or to distinct consequences of the virus on different functions of a unique cell type.

Production of IFN-{gamma}, a cytokine that is also deeply involved in the pathogenesis of endotoxin shock, was synergistically increased by infection with LDV, as it has been reported with other viruses (2–4, 6). IL-12, which appears to control LCMV-enhanced lethality and IFN-{gamma}, but not TNF production (3, 5), and which can mediate lethality in LPS-triggered shock (24) was not involved in LDV-triggered sensitization to endotoxin shock, since its induction by LPS was not increased by this virus. In contrast, LDV infection strongly enhanced IL-18 production in response to LPS challenge. Because this cytokine has been shown to have a pathogenic effect in LPS-induced shock through both IFN-{gamma}-dependent and -independent mechanisms (25, 26), and whereas its potential role has not yet been demonstrated with other viruses, this observation suggests that IL-18 might be involved in LDV-induced sensitization to endotoxin shock. Moreover, LDV was found here to delay the early IL-10 production induced by LPS, as it inhibits the secretion of other anti-inflammatory cytokines (27, 28). This depression of the early LPS-triggered IL-10 production, that has neither been found in mice infected with LCMV (2) nor with adenovirus (6), might result from the high IFN-{gamma} secretion that follows acute LDV infection (14). Although later IL-10 production was increased in LDV-infected animals, this early virally induced imbalance between pro-inflammatory cytokines, such as IL-18 and IFN-{gamma} and anti-inflammatory IL-10 might account for the enhanced response of macrophages to LPS and therefore to the increased susceptibility of mice to endotoxin shock. Thus, sensitization to LPS-induced shock by LDV differs from that by D-galactosamine, which induces sensitization of hepatocytes to TNF rather than macrophage hyperreactivity to LPS (29).

Surprisingly, type I IFNs were found to protect against LDV-exacerbated LPS shock, since mice deficient for IFN-{alpha}/ß receptor were largely more susceptible than their normal counterparts. This is in sharp contrast with a major sensitizing effect of IFN-{alpha}/ß reported after LCMV or vesicular stomatitis virus infection (4, 5). Although type I IFNs may also slightly decrease LDV replication (30, 31) the enhanced susceptibility of LDV-infected IFN-{alpha}/ß receptor-deficient mice was especially correlated with a large increase in the production of TNF, IFN-{gamma} and IL-12, but not IL-18. This observation suggests that LDV-induced IFN-{gamma} production is negatively controlled by type I IFNs, possibly through a modulation of IL-12. Such a down-regulation of IFN-{gamma} by type I IFNs through STAT1 activation has also been reported after IL-12 and anti-CD3 stimulation (32), indicating that this molecule family may have divergent effects on subsequent immune responses. Type I IFN inhibition for treatment of sepsis, as it has been suggested (33), should therefore be considered with caution since these molecules may sometimes decrease sensitivity to endotoxin, as observed here in mice infected with LDV.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Fonds National de la Recherche Scientifique (FNRS), Fonds de la Recherche Scientifique Médicale (FRSM), Loterie Nationale, Fonds Spéciaux de Recherche (UCL), the State-Prime Minister's Office - S.S.T.C. (interuniversity attraction poles, grant n°44) and the "Actions de recherche concertées" from the Communauté française de Belgique - Direction de la Recherche scientifique (concerted actions, grant no. 04/09-318), Belgium. J.-P. C. is a research director with the FNRS.


   Acknowledgements
 
The authors are indebted to P. Masson and L. Detalle for critical reading of this manuscript, to T. Briet, M.-D. Gonzales, N. Ouled Haddou, and A. Tonon for expert technical assistance.


   Abbreviations
 
LCMV, lymphocytic choriomeningitis virus
LDV, lactate dehydrogenase-elevating virus
LIF, leukemia inhibitory factor
TNF, tumor necrosis factor

   Notes
 
Transmitting editor: A. Radbruch

Received 5 December 2006, accepted 3 September 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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