JAC Advance Access originally published online on November 16, 2006
Journal of Antimicrobial Chemotherapy 2007 59(2):219-223; doi:10.1093/jac/dkl464
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Immunomodulatory effects of fosfomycin in an endotoxin model in human blood
1 Department of Clinical Pharmacology, Division of Clinical Pharmacokinetics, Medical University of Vienna Vienna, Austria 2 Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna Vienna, Austria 3 Department of Clinical Pharmacology, Division of Immunology and Hematology, Medical University of Vienna Vienna, Austria 4 Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, Medical University of Vienna Vienna, Austria
*Corresponding author. Tel: +43-1-40400-2981; Fax: +43-1-40400-2998; E-mail: claudia.marsik{at}meduniwien.ac.at
Received 29 June 2006; returned 27 July 2006; revised 18 September 2006; accepted 17 October 2006
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Abstract |
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Objectives: Although a wide range of therapeutic strategies have been developed to improve the outcome of severe sepsis, a convincing reduction in mortality is lacking. Recently, increasing attention has been paid to immunomodulatory effects of antimicrobials. This study set out to explore the immunomodulatory effects of fosfomycin, a broad-spectrum antibiotic frequently used in septic patients, at the protein and molecular levels in vitro.
Methods: Whole blood from 11 healthy volunteers was incubated with 50 pg/mL endotoxin and 100 µg/mL fosfomycin or physiological sodium chloride for 4 h. Real-time RT–PCR was performed for various pro- and anti-inflammatory cytokines. Concentrations of tumour necrosis factor (TNF)-
and interleukin (IL)-6 in the supernatant were measured using a commercially available ELISA.
Results: Incubation of human leucocytes with endotoxin increased messenger RNA (mRNA) levels of cytokines several thousand fold compared with baseline. The addition of fosfomycin significantly inhibited mRNA levels of pro-inflammatory cytokines such as IL-1-, IL-6 and TNF- after 2 h (P < 0.01), while no significant reduction was observed for the anti-inflammatory cytokines IL-4, IL-10 and IL-13 (P = 0.26). At the protein level, the concentrations of IL-6 and TNF- increased 
3000- and 600-fold after 4 h of incubation with lipopolysaccharide as compared with baseline, respectively. Addition of fosfomycin significantly reduced cytokine levels by 56% and 73% for IL-6 and TNF-, respectively.
Conclusions: Fosfomycin extensively decreased mRNA levels and release of pro-inflammatory cytokines in human blood. The broad antimicrobial coverage of fosfomycin and its immunosuppressive effects could be clinically useful in patients with sepsis.
Keywords: sepsis , in vitro , whole blood
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Introduction |
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Sepsis and septic shock are the leading cause of death in critically ill patients with lethality rates of 50–90%.1,2 Progressive systemic organ failure during sepsis has been attributed to an extensive inflammatory response accompanying the initial stages of severe sepsis.3 Lipopolysaccharide (LPS), an endotoxin which is a component of the cell wall of Gram-negative bacteria has been identified as a major trigger in this condition.4 Since the response of the host to infection is mainly mediated by inflammatory cytokines, they have become the primary focus of many studies investigating the pathophysiology of sepsis and endotoxic shock.3,5 Currently, a wide range of therapeutic strategies are under investigation in order to improve the unfavourable outcome of severe sepsis and septic shock.2,5–7 Although several of these approaches seemed initially promising, a convincing reduction of all-cause mortality is lacking and, therefore, the search for new strategies in the treatment of sepsis is ongoing.
Immunomodulatory effects of antimicrobials have been described for various classes of antibiotics including quinolones and macrolides.8–10 During the past decade, increasing attention has been paid to fosfomycin, a broad-spectrum antibiotic with excellent tissue penetration properties. Fosfomycin is approved for treatment of sepsis or severe soft tissue infection in distinct members of the European Union and has shown both favourable tissue pharmacokinetics and antimicrobial activity in sepsis.11,12 Recently, it has been demonstrated that administration of fosfomycin to mice during Pseudomonas aeruginosa induced sepsis impairs substantially the acute inflammatory response.13 This was confirmed by animal and in vitro studies indicating that fosfomycin modulates the production of pro- and anti-inflammatory cytokines induced by LPS.14,15
However, LPS concentrations used in these studies were 2500–50 000 times higher compared with median LPS levels observed during Gram-negative sepsis in vivo and, therefore, are unlikely to represent conditions in septic ICU patients.14–16 In addition, previous studies were limited to investigation of some selected cytokines on protein levels by ELISA, which cannot provide information about the modifications of the network pattern of pro- and anti-inflammatory cytokines on the molecular level.
Thus, this study employed an in vitro endotoxin model to explore the immunomodulatory effects of fosfomycin in human blood both at the protein and molecular levels. In addition, we investigated levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-, the most important pro-inflammatory cytokines, by ELISA. We used a highly sensitive real-time PCR (RT–PCR) for quantitative analysis of messenger RNA (mRNA) encoding for pro- and anti-inflammatory cytokines. Measuring cytokine response at the mRNA level allows for detection of rapid interactions of fosfomycin with the DNA and allows the detection of small changes of cytokine response to stimulation by endotoxin. This is the first time that RT–PCR has been used to detect effects of antibiotics on cytokine response in a sepsis model.
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General
The study took place at the Department of Clinical Pharmacology, Medical University of Vienna, Austria. The protocol was approved by the local ethics committee and was performed in accordance with the Declaration of Helsinki, current revisions of the Good Clinical Practice (GCP) Guidelines of the European Commission and the Good Scientific Practice guidelines of the Medical University of Vienna.
Study subjects
Eleven healthy male volunteers participated in this trial. All healthy volunteers had normal laboratory values and none of them had regular concomitant medication within the last 2 weeks prior to the start of study. Written informed consent was obtained prior to the collection of blood samples.
Blood sampling and incubation with study drugs
Blood samples were collected by venipunctures into Vacutainer tubes (Becton Dickinson, Vienna, Austria) anticoagulated with EDTA. In brief, immediately after collecting of blood samples, 900 µL of EDTA anticoagulated blood was incubated either blank, with LPS (Endotoxin Escherichia coli 026:B6, Sigma Aldrich, Vienna, Austria) at 50 pg/mL and physiological sodium chloride (Natrium chloratum physiologicum 0.9%, Meditrade Medipharm, Kufstein, Austria) or with LPS at 50 pg/mL and fosfomycin at 100 µg/mL (Sandoz, Kundl, Austria) for 4 h at 37°C.
Messenger RNA isolation and transcription
Blood of five randomly chosen subjects was investigated at the molecular level after 2 and 4 h. The QuiAmp Blood RNA easy kit (Qiagen, Valencia, USA) was used to isolate cellular mRNA according to instructions. Red blood cells were selectively lysed and leucocytes were collected by centrifugation. Leucocytes were then lysed using highly denaturating conditions which immediately inactivate RNAses and enzyme inhibitors. The QuiAmp procedure completely removes contaminants, yielding high-quality RNA suitable for RT–PCR.
Subsequently, the mRNA was directly transcribed into cDNA using a reverse transcription kit (Applied Biosystems, Foster City, CA, USA) as described previously and was stored at –80°C until further analysis.17
To avoid false-positive amplifications we used AmpErase® (Roche Diagnostics Corporation, Indianapolis, USA) uracil-N-glycosylase (UNG), a pure, nuclease-free, 26 kDa recombinant enzyme encoded by the E. coli UNG gene. UNG acts on single- and double-stranded dU-containing DNA by hydrolysing uracil-glycosidic bonds at dU-containing DNA sites. The enzyme causes the release of uracil, thereby creating an alkali-sensitive apyrimidic site in the DNA. The enzyme has no activity on RNA or dT-containing DNA.18
Real-time PCR (RT–PCR)
For quantification of mRNA the ABI PRISM® 7700 System (Applied Biosystems, Foster City, USA) was employed according to instructions. In brief, each sample was introduced into a TaqMan® Human Cytokine Card which contains probes and primers for 24 cytokine targets as well as the 18S ribosomal RNA control. The sample was introduced via an attached filling reservoir and a vacuum loading process using the ABI PRISM® Card Filling Station. For reading, the TaqMan® Human Cytokine Cards were loaded into the ABI PRISM® 7700 Sequence Detection System which allows for cycle-by-cycle detection of the increase in PCR product.
mRNA of 24 different cytokines was quantified in this study: IL-1-, IL-1-ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p35, IL-12p40, IL-13, IL-15, IL-17, IL-18, G-CSF, GM-CSF, M-CSF, IFN-
, LT-ß, TGF-ß, TNF-, TNF-ß (Abbreviations: G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte—macrophage colony stimulating factor; M-CSF, macrophage colony stimulating factor; IFN, interferon; LT, lymphotoxin; TGF, transforming growth factor). 18S was used as a housekeeping gene for multiplexing because of its stable expression under LPS stimulus (C. Marsik and B. Jilma, unpublished data). Target genes were normalized against their reference gene (18S) and data were expressed as relative fold increase or decrease from baseline values.19
Relative mRNA expression was determined by the 
CT method by Livak and Schmittgen as described previously.20 The CT numbers indicate the difference in amplification cycles between target and housekeeping (18S) gene. Results were only included in the final analysis if at least 5-fold increase in mRNA levels compared with baseline was observed to allow for reliable data analysis.
ELISA for IL-6 and TNF-
For determination of IL-6 and TNF- levels a commercially available ELISA (Quantikine®, R & D systems, Minneapolis, USA) was used according to the instruction of the manufacturer. Concentrations of TNF- and IL-6 levels in the supernatant of blood of all 11 subjects were measured at baseline, 2 and 4 h after incubation with study drugs. Upper limit of quantification was 2000 pg/mL for TNF- and 3000 pg/mL for IL-6. If concentrations were beyond the upper limit of detection, the value of the detection limit was used for calculations as this was considered most conservative.
Statistical calculations
All statistical calculations were performed using commercially available statistical software (Statistica®, StatSoft, Inc., Tulsa, USA). Data in the text are expressed as means ± SEM. Statistical comparisons were done by the use of Wilcoxon matched-pairs test. A P value <0.05 was considered significant. For mRNA, number of useable pairs for statistical comparison (=at least 5-fold increase in mRNA levels as compared with baseline for simulations both with and without the addition of fosfomycin) is indicated in brackets.
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Real-time PCR
After 2 h incubation of human leucocytes with 50 pg/mL endotoxin mRNA levels of cytokines increased several thousand-fold up to a peak value of 17 947 ± 8292 compared with baseline. However, the addition of 100 µg/mL fosfomycin reduced the increase of mRNA significantly (P < 0.0005) to levels of 3744 ± 1675-fold. After 4 h of incubation with endotoxin increase of cytokine levels compared with baseline was comparable for stimulations with or without the addition of fosfomycin presenting values of 3933 ± 1485 and 4219 ± 1466-fold, respectively (P = 0.12).
To investigate whether there might be a difference between mRNA levels of pro- and anti-inflammatory cytokines an additional analysis was performed for the sum of the early pro-inflammatory cytokines IL-1-, IL-6 and TNF- and the major anti-inflammatory cytokines IL-4, IL-10 and IL-13.1,21 Similar to the sum of all investigated cytokines, the addition of fosfomycin significantly reduced mRNA levels of the early pro-inflammatory cytokines after 2 h of incubation with LPS (P < 0.01) while no reduction was detectable after 4 h (P = 0.77). In contrast, for anti-inflammatory cytokines neither after 2 (P = 0.26) nor after 4 (P = 0.57) h was a significant reduction of mRNA levels observed by the addition of fosfomycin.
Figure 1 depicts mRNA levels after the stimulation of leucocytes with LPS for selected cytokines. The effect of fosfomycin is presented relative to the effects of LPS mono-treatment (indicated by the horizontal line) after 2 and 4 h of incubation.
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ELISA IL-6 and TNF-
Concentrations of IL-6 and TNF- in the supernatant after 4 h of incubation with LPS increased 3000- and 600-fold, respectively, as compared with baseline. Absolute concentrations of IL-6 and TNF- in the supernatant after incubation of whole blood over a period 2 and 4 h with LPS alone or LPS and fosfomycin are depicted in Figure 2. After 2 h no significant difference in the cytokine concentrations between incubation with LPS alone and incubation with LPS and fosfomycin was detected. After 4 h of incubation, however, the addition of fosfomycin significantly reduced cytokine levels by 56% and 73% for IL-6 and TNF-, respectively.
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Discussion |
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Several therapeutic strategies including anti-inflammatory agents, drugs modifying coagulation and antibodies against endotoxin or specific cytokines have been investigated for their ability to decrease the overwhelming inflammatory response associated with sepsis.2,5–7 However, besides corticosteroids at low doses and activated protein C in case of severe sepsis, most of these therapeutic approaches were not able to reduce mortality markedly,6 but were often associated with the increased risk of side effects.22–24 Difficulties in developing successful therapeutic interventions for this condition have been attributed to insufficient knowledge on the timing of mediator release and the complex cytokine balance during sepsis.5 In addition, inhibiting the host inflammatory response without detailed knowledge about modifications at the protein and molecular level may not be beneficial without disadvantage because immune cells and cytokines play both pathogenic and protective roles and the balance between pro- and anti-inflammatory reactions is critical for clinical outcome in septic patients.1,5
Since broad-spectrum antibiotics are routinely administered in the condition of sepsis, choosing an antibiotic with potency to modify anti-inflammatory response would lack additional side effects and would more likely result in a positive risk to benefit relationship than other approaches. In this study, therefore, the previously observed effect of fosfomycin to modulate inflammatory response was characterized at the molecular level for a wide range of cytokines at 2 and 4 h. This approach allows more insight with respect to the influence on transcription and release of mediators at the initial stage of sepsis.13–15 Since previous studies have described that the immune-modulatory effect of fosfomycin is concentration dependent,14,15,25 in this study fosfomycin was used at a final concentration of 100 µg/mL, i.e. a concentration that has been shown to be achieved in plasma and soft tissues after intravenous administration of standard doses of fosfomycin of 4 or 8 g.11 While previous in vitro studies employed LPS concentrations of up to 100 ng/mL for the induction of inflammatory response, the much lower concentration of 50 pg/mL was chosen for this study as this represents conditions during Gram-negative sepsis in vivo.14,16 In addition, incubation of whole blood instead of the previously predominantly used cell line model has been performed in this study in an attempt to design a more realistic in vitro model.
At mRNA level, the addition of fosfomycin significantly inhibited the LPS-induced expression of the pro-inflammatory cytokines IL-1-, IL-6 and TNF- after 2 h (P < 0.01), while at the same time no significant reduction of mRNA levels was observed for the anti-inflammatory cytokines IL-4, IL-10 and IL-13 (P = 0.26). In contrast, after 4 h of endotoxin incubation mRNA levels of neither pro- nor anti-inflammatory cytokines had been significantly decreased by fosfomycin. This could be explained by negative feedback mechanisms induced by the extensive release of IL-1-, IL-6 and TNF-, leading to consecutive down-regulation of transcription of pro-inflammatory cytokines independent of the fosfomycin effect.
At the protein level, after 4 h of incubation with LPS a dramatic increase in IL-6 and TNF- of 3000- and 600-fold, respectively, was observed as compared with baseline. While after 2 h no significant difference in the cytokine concentrations between incubation with LPS alone and incubation with LPS and fosfomycin was observed, after 4 h of incubation, the addition of fosfomycin had significantly reduced cytokine levels by 56% and 73% for IL-6 and TNF-, respectively. The fact that fosfomycin reduces protein levels of IL-6 and TNF- in the supernatant after 4 h but not after 2 h can be explained by the results at mRNA level discussed above. The significant difference in mRNA levels of pro-inflammatory cytokines observed at 2 h has subsequently induced delayed differences in cytokine production and release at 4 h.
The ability of fosfomycin to decrease the release of IL-6 and TNF- observed in this study is in good agreement with a previous finding from Matsumoto et al.13 using P. aeruginosa to induce sepsis in mice. In contrast, TNF- levels were demonstrated to fall by the addition of fosfomycin in previous in vitro studies using LPS-stimulated human monocytes. In addition, a dose-dependent increase in IL-6 levels was observed by adding fosfomycin.14 This discrepancy might be due to the use of native blood in this study while isolated monocytes were investigated by others. The use of monocytes, however, removes the opportunity to identify possible interactions with other blood cells.26 Another methodological difference is based on the 2000-fold higher concentration of LPS used to stimulate monocytes in the study by Morikawa et al. This high concentration of LPS might have altered the production and release of IL-6 to an unknown extent, which is in contrast to our study designed to present appropriate real-life conditions during sepsis. However, it must be pointed out that comparison of the present findings with previously published results on immunomodulatory effects of fosfomycin cannot consider all methodological differences. Possible explanations may include different concentrations of LPS, the type of stimulated cells, the time of exposure to LPS and different bacterial origin of LPS. Thus, a direct comparison of results derived from different in vitro models should be done only with caution.
Although the classification of cytokines as pro- or anti-inflammatory cytokines is widely accepted throughout the literature, it has to be noticed that this common clear-cut classification is a simplification.1 The net effect of an inflammatory response is determined by the balance between pro- and anti-inflammatory cytokines and the type, duration and extent of cellular activities induced by one particular cytokine can be influenced considerably by the nature of the target cells and its preconditions. IL-6, for example, may have pro- and anti-inflammatory properties depending on the stage of inflammation.21 In addition, the clinical impact of a reduction of cytokine levels by 56% and 73% as observed for IL-6 and TNF- together with the fact that mRNA levels of pro-inflammatory cytokines are suppressed, while mRNA levels of anti-inflammatory cytokines remain unaffected by the addition of fosfomycin is currently unknown. Although both limitations apply for all in vitro studies investigating cytokines, the fact that our in vitro approach focused on the determination of early cytokine response after 2 and 4 h qualifies this study to demonstrate suppression of early inflammatory response by fosfomycin more precisely than previous studies.
In this study, a clinically relevant concentration of fosfomycin was chosen. All experiments were performed exclusively at a concentration of 100 µg/mL, although fosfomycin follows linear elimination at the ß-phase. Therefore, the effect of dynamic changes of fosfomycin concentrations over time on immunomodulatory effects was not evaluated in this study. Another limitation was that the effect of fosfomycin was tested in vitro at situations mimicking only a very early state of sepsis. Thus, further studies exploring the dynamic effect of fosfomycin and testing its effect at a more progressed stage of sepsis should be performed.
In conclusion, this study showed that fosfomycin at clinically achieved serum concentrations significantly reduces the peak of mRNA levels of pro-inflammatory cytokines after 2 h of endotoxin exposure while anti-inflammatory cytokines remain unaffected. The reduction of mRNA levels of pro-inflammatory cytokines by fosfomycin was reflected by the subsequent decrease in protein levels of IL-6 and TNF- after 4 h. Thus, we have for the first time demonstrated that the immunomodulatory effect of fosfomycin is rather based on the reduction of the transcription of pro-inflammatory cytokines than on inhibition of cytokine release. These data provide circumstantial evidence that fosfomycin is potentially able to reduce inflammatory response at the initial states of endotoxaemia in septic patients, particularly in those who show overwhelming inflammatory reaction. Nevertheless, in vivo studies are desirable to confirm the clinical impact of the present findings.
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None to declare.
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References |
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