Immune dysfunction in trauma patients is associated with immune system activation and inflammation. The cytokine-inducible enzyme IDO (indoleamine 2,3-dioxygenase) initiates the degradation of the essential aromatic amino acid tryptophan via the kynurenine pathway and could contribute to deficient immune responsiveness. Activated IDO is indicated by an increased kyn/trp (kynurenine/tryptophan) ratio. The aim of the present study was to investigate whether tryptophan degradation is associated with outcome in patients post-trauma. Tryptophan and kynurenine concentrations were measured by HPLC in serum specimens of 15 patients post-trauma during 12–14 days of follow-up. Up to five samples within this observation period from each patient were included in this analysis, and a total a 69 samples were available. For further comparisons, concentrations of the immune activation marker neopterin were measured. Compared with healthy controls, the average kyn/trp ratio and kynurenine concentrations were increased in patients, whereas tryptophan concentrations were decreased. During follow-up, increased kyn/trp ratio and kynurenine concentrations (all P<0.001) were observed, whereas the changes in tryptophan concentrations were not significant. Non-survivors had higher kyn/trp ratios and kynurenine concentrations compared with survivors. The kyn/trp ratio correlated with neopterin concentrations (rs=0.590, P<0.001). In conclusion, these results imply that increased tryptophan degradation in patients is due to activated IDO, which most probably is a consequence of a host defence response. These findings support a possible role for IDO in the development of immunodeficiency and death in patients.

INTRODUCTION

The inflammatory response in trauma patients is crucial for their fate. In such patients, significant activation of various immune system compartments is demonstrated which, however, is accompanied by diminished functional immune response and immune paralysis [1]. The enzyme IDO (indoleamine 2,3-dioxygenase) converts tryptophan into kynurenine and is strongly inducible by pro-inflammatory stimuli and, preferentially, by the cytokine IFN-γ (interferon-γ) [2,3]. Activated IDO decreases the availability of the essential amino acid tryptophan and is considered as an immune defence mechanism, which suppresses the growth of intracellular bacteria, viruses and parasites, such as toxoplasma, and malignant tumour cells [35]. However, T-cell proliferation can also be inhibited by IDO activity [6] and, thus, the immune response may be suppressed when tryptophan levels are decreased. In patients after multiple trauma, tryptophan deficiency has been found to be associated with the decline of lymphocyte numbers [7].

Pro-inflammatory stimuli such as the Th1-type cytokine IFN-γ induce IDO and neopterin production in human monocyte-derived macrophages and DCs (dendritic cells) [8,9]. In several groups of patients, for example suffering from virus infections, autoimmune syndromes and also arteriosclerosis and cancer, enhanced tryptophan degradation concurs with increased neopterin formation [1013]. In trauma patients, plasma concentrations of neopterin are able to predict outcome [1,14,15]. From the results, it would be expected that, in multiple trauma patients, increased neopterin production will be paralleled by activation of IDO, which could represent a key to understanding the negative contribution that immune activation and IFN-γ production might have in trauma patients.

In the present study, concentrations of tryptophan and kynurenine and the kyn/trp ratio (kynurenine/tryptophan ratio) were analysed in trauma patients and compared with outcome. In addition, results on tryptophan metabolism were compared with concentrations of neopterin.

MATERIALS AND METHODS

Patients

A total of 15 patients post-trauma were included in the present study who either were admitted to the ICU (Intensive Care Unit) of the Medical University of Vienna or to the ICU of Lorenz Boehler Trauma Center Vienna (Table 1). Inclusion criteria were age between 18 and 80 years and had evidence of a multiple trauma [ISS (Injury Severity Score) >16). Exclusion criteria were immunosuppressive therapy, HIV infection or any other chronic disease. All patients were otherwise healthy before the trauma. The average length of stay in the ICU was 25.3±20.1 days. During follow-up, six patients died on days 7, 10, 14, 17, 26 and 37. Serum samples were collected every third day during 12–14 days of follow-up. For statistical analyses, samples were collected from each patient and divided into five groups depending on the day of collection: days 1–2, group I; days 3–5, group II; days 6–8, group III; days 9–12, group IV; and days 13–14, group V. In total, every patient contributed 3–5 samples to the total number of 69 samples analysed, which corresponds to six missing specimens over the whole period. All patients received standard parenteral nutrition [1700 kcal/day (where 1 kcal≈4.184 kJ) and 100 g of amino acids/day] after the end of hypodynamic shock.

Table 1
Characteristics of the all of the patients and when subdivided into survivors and non-survivors

Values are means±S.D. Determined using a Student's t test* or Pearson's χ2 test†.

All patientsSurvivorsNon-survivorsP value
n 15  
Age (years) 40.4±17.2 34.3±10.4 50.3±21.8 0.141* 
Gender (n) (female/male) 3/12 2/7 1/5 0.605† 
Length of stay in ICU (days) 25.3±20.1 32.9±21.3 14.0±12.1 0.137* 
Received inotropes (n) (yes/no) 14/1 8/1 6/0 0.412† 
Apache score 17.5±6.5 15.0±4.7 20.8±7.3 0.154* 
ISS 39.1±13.1 35.2±10.9 44.8±14.9 0.170* 
All patientsSurvivorsNon-survivorsP value
n 15  
Age (years) 40.4±17.2 34.3±10.4 50.3±21.8 0.141* 
Gender (n) (female/male) 3/12 2/7 1/5 0.605† 
Length of stay in ICU (days) 25.3±20.1 32.9±21.3 14.0±12.1 0.137* 
Received inotropes (n) (yes/no) 14/1 8/1 6/0 0.412† 
Apache score 17.5±6.5 15.0±4.7 20.8±7.3 0.154* 
ISS 39.1±13.1 35.2±10.9 44.8±14.9 0.170* 

Samples from 49 healthy blood donors (21 women and 28 men; age, 35.2±13.5 years) served as a reference group [16].

The study was performed according to the Declaration of Helsinki (2000). The protocol was approved by the local ethics committee, and written informed consent was granted by the next of kin. The observed values of all of the study parameters had no influence on the course of therapy.

Measurements

Concentrations of tryptophan and kynurenine were determined by HPLC as described previously [16,17]. The kyn/trp ratio was calculated and expressed as μmol/l kynurenine per mmol/l tryptophan. Concentrations of neopterin were measured using a commercially available ELISA (BRAHMS; detection limit of 2 nmol/l), according to the manufacturer's instructions.

Statistical analysis

Demographic parameters were compared using a Student's t test or χ2 test. Results are expressed as means±S.D. Because not all of the data sets had a normal distribution, non-parametric statistics were applied for data analyses. A Kruskal–Wallis H test was used to determine differences in the median values between the five time groups, and a Mann–Whitney U test was subsequently applied for direct comparisons between grouped data. Spearman's rank correlation coefficients (rs) were calculated for regression analyses. Changes during follow-up were calculated using a paired rank test. Repeated-measures ANOVA with five steps was calculated for the 5 days of measurement. SPSS (version 14) was used. P values<0.05 were considered to indicate significant differences.

RESULTS

The kyn/trp ratio and concentrations of kynurenine were increased and concentrations of tryptophan were decreased in patients compared with the reference group (Figure 1). None of the parameters correlated with the Apache score of patients. During follow-up, significant changes in the kyn/trp ratio (H=21.3, P<0.001) and kynurenine concentrations (H=21.2, P<0.001) were observed (Figure 1). The kyn/trp ratio and kynurenine concentrations increased from group I to II and remained higher than baseline throughout the further follow-up period (kyn/trp ratio, P<0.01, P<0.01, P<0.01 and P<0.06 for groups II, III, IV and V compared with group I respectively; kynurenine, P<0.01, P<0.01, P<0.01 and P<0.06 for groups II, III, IV and V compared with group I respectively). Concentrations of tryptophan did not significantly differ from day 1 throughout the study period.

Serum kyn/try ratio (top panel), kynurenine concentrations (middle panel) and tryptophan concentrations (bottom panel) in plasma samples of 15 patients post-trauma during follow-up for 2 weeks

Figure 1
Serum kyn/try ratio (top panel), kynurenine concentrations (middle panel) and tryptophan concentrations (bottom panel) in plasma samples of 15 patients post-trauma during follow-up for 2 weeks

The individual values for the nine survivors are indicated by the solid line, with the mean value for each group indicated by the black box with white cross. The individual values for the six non-survivors are indicated by the dotted lines, with the mean value for each group represented by the black triangle. The mean values for the whole group of patients is indicated by the black circles. The grey shading represents the range of control values from a reference group of healthy subjects. The numbers of subjects (survivors/non-survivors) is shown in each group. *P<0.05 compared with non-survivors.

Figure 1
Serum kyn/try ratio (top panel), kynurenine concentrations (middle panel) and tryptophan concentrations (bottom panel) in plasma samples of 15 patients post-trauma during follow-up for 2 weeks

The individual values for the nine survivors are indicated by the solid line, with the mean value for each group indicated by the black box with white cross. The individual values for the six non-survivors are indicated by the dotted lines, with the mean value for each group represented by the black triangle. The mean values for the whole group of patients is indicated by the black circles. The grey shading represents the range of control values from a reference group of healthy subjects. The numbers of subjects (survivors/non-survivors) is shown in each group. *P<0.05 compared with non-survivors.

Patients who died during follow-up had a significantly higher kyn/trp ratio (P<0.05 in groups II, IV and V compared with survivors; Figure 1) and higher kynurenine concentrations (P<0.05 in group IV compared with survivors). Tryptophan concentrations were lower in nonsurvivors compared with survivors, but did not differ significantly. When the concentrations measured in nonsurvivors were compared with baseline, the increase in the kyn/trp ratio and kynurenine became significant in group III (P<0.05), and the decrease in tryptophan approached significance in group V (U=1.82, P=0.068). Repeated-measures ANOVA showed a significant difference in kynurenine concentrations (P<0.05) and the kyn/trp ratio (P<0.01) between survivors and non-survivors.

Mean concentrations of the marker neopterin (19.7±21.7 nmol/l) were increased in patients compared with the reference group. Patients who died during follow-up had higher neopterin concentrations (P<0.05 in groups II and III, and P<0.01 in groups IV and V) compared with survivors. In the whole data set, a positive correlation between the kyn/trp ratio and neopterin concentrations (rs=0.590, P<0.001; results not shown) was observed.

DISCUSSION

The results of the present study show that tryptophan metabolism is altered in patients suffering from trauma compared with healthy controls. Compared with healthy controls, kynurenine concentrations and the kyn/trp ratio were increased in patients, whereas tryptophan concentrations were decreased. During follow-up, a higher rate of kynurenine accumulation and tryptophan degradation, as indicated by the increased kyn/trp ratio, was observed in non-surviving trauma patients compared with survivors. These findings confirm and extend earlier observations on enhanced degradation of tryptophan by IDO in an independent set of patients after major trauma [7].

In parallel with the increase in kynurenine concentrations and the kyn/trp ratio, an increase in neopterin formation was observed. Because the increase in neopterin is associated with Th1-type cytokines, such as IFN-γ [18], which induces both tryptophan degradation via IDO and neopterin production in parallel [8,11], we assume that accelerated tryptophan degradation is due to enhanced IDO activity in these patients. Low tryptophan concentrations are unlikely to be related to a reduced dietary intake of this essential amino acid, because, in this case, a decrease in kynurenine and no change in the kyn/trp ratio would be expected. Increased IDO activity may indeed account for lowered tryptophan concentrations and, because of the significant association between neopterin concentrations and the kyn/trp ratio, tryptophan degradation in our patients also appears likely to be induced by pro-inflammatory stimuli, of which IFN-γ is the strongest inducer of IDO in macrophages and DCs.

Measurement of circulating IFN-γ concentrations in serum/plasma of patients is usually very insensitive, which is why we refrained from analysing these in our patients. As with other cytokines, IFN-γ rapidly binds to its specific receptors on target cells or their shed soluble forms and, therefore, the low concentrations in the blood limit the diagnostic application.

Post-trauma, immunocompetent cells appear to respond against non-self structures, and cells produce cytokines, including IFN-γ, in an attempt at halting the growth of pathogens. Activation of IDO as one out of several important antimicrobial mechanisms triggered by IFN-γ, however, not only affects microbes, but it potentially may also counteract the growth and development of T-cells [6]. An equilibrium might develop between the degree of activation of immunocompetent cells, their suppressive effect on microbes and also on themselves. In some patients, the bactericidal effect of IDO will be able to stop the infectious process; in others, immunocompetent cells will be affected by IDO activity more than the microbes and, thus, the depletion of T-cells and immunosuppression will result from the host's immune response against invading pathogens [19,20]. Certainly the present study is too preliminary to give a definitive answer to this question.

A similar relationship between enhanced tryptophan degradation and immune activation has been found previously in several other chronic diseases, such as HIV-1 infection or malignancy [11,2124]. Moreover, in patients with HIV-1 infection and with cancer, enhanced tryptophan degradation and increased neopterin production were found to strongly predict shortened survival [2123]. In addition, in patients with HIV-1 infection, the antimicrobial/antiviral activity of IDO is suggested to contribute to immunodeficiency [24,25] and may explain why immune activation markers are strong predictors of outcome. From in vitro studies, it was concluded that not only the decline in tryptophan concentrations, but also the increase in formation of toxic tryptophan catabolites could be involved in the development of T-cell unresponsiveness [26]. Interestingly in our present study, only an increase in kynurenine concentrations, but not the decline in tryptophan, was significantly associated with the outcome of the patients. Thus findings may favour a role of tryptophan catabolites, rather than tryptophan lowering, in immune system deviations which might be of relevance for the fatal outcome of some of our patients. Pellegrin et al. [7] have shown previously that a decline in tryptophan was associated with the fall in T-lymphocytes in patients after trauma [7]. In our present study, the decrease in tryptophan concentrations did not reach the level of significance. The standard parenteral nutrition regimen, which contains tryptophan as well as other essential amino acids and was initiated early in our patients, may have counteracted to some degree the loss of tryptophan, despite accelerating degradation of the amino acid further and increasing kynurenine concentrations further.

Tryptophan deficiency resulting from its accelerated catabolism could also be involved in the increased risk of developing anaemia, cachexia and neuropsychiatric abnormalities [11,27,28]. Nevertheless, the relevance of this assumption needs to be clarified in further extended studies.

In conclusion, accelerated tryptophan degradation was found in non-surviving patients post-trauma. The same was true for higher concentrations of neopterin, which confirms and extends previous studies [14,15]. Tryptophan degradation may represent one important aspect in the development of the post-traumatic failure of the immune system to respond appropriately. More extended and follow-up studies examining the impact of tryptophan degradation and its relationship with cellular immune activation in patients suffering from trauma may provide interesting new findings, which should be helpful in defining new therapeutic intervention strategies. Larger clinical studies are necessary to find out more about the potential prognostic expressiveness of tryptophan metabolism in patients suffering from trauma.

FUNDING

This work was supported by Stiftung Propter Homines, Vaduz -Fürstentum Liechtenstein.

We thank Miss Astrid Haara for excellent technical assistance.

Abbreviations

     
  • DC

    dendritic cell, ICU, Intensive Care Unit

  •  
  • IDO

    indoleamine 2,3-dioxygenase

  •  
  • IFN-γ

    interferon-γ

  •  
  • ISS

    Injury Severity Score

  •  
  • kyn/trp ratio

    kynurenine/tryptophan ratio

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