Both endothelial and immune dysfunction contribute to the high mortality rate in human sepsis, but the underlying mechanisms are unclear. In response to infection, interferon-γ activates indoleamine 2,3-dioxygenase (IDO) which metabolizes the essential amino acid tryptophan to the toxic metabolite kynurenine. IDO can be expressed in endothelial cells, hepatocytes and mononuclear leukocytes, all of which contribute to sepsis pathophysiology. Increased IDO activity (measured by the kynurenine to tryptophan [KT] ratio in plasma) causes T-cell apoptosis, vasodilation and nitric oxide synthase inhibition. We hypothesized that IDO activity in sepsis would be related to plasma interferon-γ, interleukin-10, T cell lymphopenia and impairment of microvascular reactivity, a measure of endothelial nitric oxide bioavailability. In an observational cohort study of 80 sepsis patients (50 severe and 30 non-severe) and 40 hospital controls, we determined the relationship between IDO activity (plasma KT ratio) and selected plasma cytokines, sepsis severity, nitric oxide-dependent microvascular reactivity and lymphocyte subsets in sepsis. Plasma amino acids were measured by high performance liquid chromatography and microvascular reactivity by peripheral arterial tonometry. The plasma KT ratio was increased in sepsis (median 141 [IQR 64–235]) compared to controls (36 [28–52]); p<0.0001), and correlated with plasma interferon-γ and interleukin-10, and inversely with total lymphocyte count, CD8+ and CD4+ T-lymphocytes, systolic blood pressure and microvascular reactivity. In response to treatment of severe sepsis, the median KT ratio decreased from 162 [IQR 100–286] on day 0 to 89 [65–139] by day 7; p = 0.0006) and this decrease in KT ratio correlated with a decrease in the Sequential Organ Failure Assessment score (p<0.0001). IDO-mediated tryptophan catabolism is associated with dysregulated immune responses and impaired microvascular reactivity in sepsis and may link these two fundamental processes in sepsis pathophysiology.
Citation: Darcy CJ, Davis JS, Woodberry T, McNeil YR, Stephens DP, et al. (2011) An Observational Cohort Study of the Kynurenine to Tryptophan Ratio in Sepsis: Association with Impaired Immune and Microvascular Function. PLoS ONE 6(6): e21185. doi:10.1371/journal.pone.0021185
Editor: Jane Deng, University of California Los Angeles, United States of America
Received: November 30, 2010; Accepted: May 23, 2011; Published: June 22, 2011
Copyright: © 2011 Darcy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was funded by the National Health and Medical Research Council of Australia (Program Grants 290208, 496600; Practitioner Fellowship to NMA, Scholarship to JSD) and an Australian Postgraduate Award to CJD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Sepsis is a systemic inflammatory response to infection . Despite advances in its management, severe sepsis still has a mortality rate of 30–50% , , . Both immune and endothelial dysfunction are thought to contribute to the high mortality rate in sepsis , , however the underlying mechanisms are not completely understood.
Tryptophan is an essential amino acid that is central to cellular respiration  and neurotransmission , and is a key immune mediator. During inflammation, tryptophan is metabolised by indoleamine 2,3-dioxygenase (IDO) to the toxic metabolite kynurenine. IDO activity is measured by the ratio of kynurenine to tryptophan (the KT ratio). Endothelial cells, monocytes, renal tubular epithelial cells and hepatocytes express IDO in response to interferon-γ , , , ,  and IL10 stabilises IDO expression .
IDO activity regulates a number of immune responses. Increased IDO activity inhibits T cell function  and proliferation , ,  and contributes to T cell apoptosis . Furthermore, elevated IDO activity inhibits nitric oxide synthase and vice versa , , . Recent isotope studies have shown that systemic NO production is either reduced or unchanged in human sepsis compared with healthy controls , , .
In addition to regulating the immune response, IDO activity may also regulate endothelial function. Kynurenine, a metabolite of IDO, has recently been described as an endogenous vasorelaxing factor . Increased IDO activity would therefore be expected to directly decrease systemic vascular resistance. Additionally, as IDO inhibits NOS, IDO may indirectly affect endothelial function by impairing NO-dependent microvascular reactivity. NO is essential for normal endothelial function and NO-dependent microvascular reactivity has been previously shown to be impaired in patients with sepsis, in proportion to disease severity , . Finally, plasma kynurenine concentrations have been associated with markers of endothelial dysfunction in patients with end-stage renal disease .
IDO activity correlates with disease severity in patients with chronic inflammatory diseases such as human immunodeficiency virus , systemic lupus erythematosus  and malignancy , but little is known about IDO activity in acute inflammatory states. A raised KT ratio has recently been reported in patients with bacteremia .
We investigated the relationship between the KT ratio and disease severity in sepsis. We hypothesised that the KT ratio would be related to IFN-γ and IL10 concentrations, and inversely related to both T cell lymphopenia and microvascular reactivity, a measure of endothelial NO bioavailability.
We evaluated patients with sepsis and hospital controls who were part of a previously reported study of endothelial function in sepsis . Sepsis patients had suspected or proven infection and the presence of two or more criteria for the systemic inflammatory response syndrome (SIRS) within the last 4 hours . Severe sepsis patients had organ dysfunction or shock at the time of enrolment according to the American College of Chest Physicians/Society of Critical Care Medicine criteria , . Sepsis severity was estimated using the Acute Physiology and Chronic Health Evaluation (APACHE) II score from the first 24 hours of admission and daily modified Sequential Organ Failure Assessment (SOFA) score . Patients were enrolled within 24 hours of ICU admission or within 36 hours of ward admission. Control subjects were recruited from hospital patients who had not met SIRS criteria within the last 30 days and who had no clinical or laboratory evidence of inflammation or infection. Written informed consent was obtained from all participants or next of kin. All sepsis patients had undergone resuscitation and were haemodynamically stable at the time of study enrolment. The study was approved by the Human Research Ethics Committee of Menzies School of Health Research and the Department of Health and Community Services.
Blood collection and lymphocyte counts
Venous blood was collected in lithium heparin tubes at enrolment, day 2–4, and day 7 until discharge from the hospital or death. Whole blood differential white cell counts were measured by Coulter Counter. Lymphopenia was defined as an absolute lymphocyte count less than 1.2×103/µL . Plasma was separated and stored at −80°C.
Lymphocytes were analysed in more detail in a subset of patients from whom samples were processed within 30 minutes of collection, matched for age and gender. Peripheral blood mononuclear cells were separated using Ficoll-Paque™ Plus (GE Healthcare Biosciences, Uppsala, Sweden) and cryopreserved in fetal calf serum and dimethyl sulfoxide. Cells were thawed and stained with appropriate antibodies and analysed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, MA, USA). Antibodies were sourced from Biolegend, California, USA (CD3, CD16 and CD56) or BD Biosciences Pharmingen, California, USA (CD4 and CD8). Results were analysed using Flow Jo software (Tree Star, Oregon, USA). T cells were defined as CD3+ lymphocytes and natural killer cells were defined as CD3−CD16+CD56+ lymphocytes.
Tryptophan and kynurenine measurements
Plasma tryptophan and kynurenine concentrations were measured by High Pressure Liquid Chromatography (HPLC; Shimadzu, Kyoto, Japan) with UV (250 nm) and fluorescence (excitation 250 nm, emission 395 nm) detection, using a method modified from van Wandelen and Cohen . The kynurenine to tryptophan (KT) ratio was calculated by dividing the kynurenine concentration (µmol/L) by the tryptophan concentration (µmol/L) and multiplying the quotient by 1000 , , .
Plasma cytokine measurements
Concentrations of plasma IFN-γ, IL6 and IL10 were determined using a cytometric bead array (Human Th1/Th2 Cytokine Kit II, BD Biosciences Pharmingen, CA, USA) and a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, MA, USA). Results were analysed using FCAP array version 1.0.1 (Soft Flow Hungary for Becton Dickinson Biosciences). The lower limits of detection (LLD) of the assay were 2.5 pg/mL for IFN-γ and 10 pg/mL for IL6 and IL10. Values below the LLD were assigned a value halfway between zero and the LLD for statistical analysis. Cytokines were only measured if plasma had been frozen within 2 hours of collection.
Measurement of endothelial function
Sepsis patients underwent serial bedside reactive hyperemia peripheral arterial tonometry (RH-PAT) measurements at enrolment, day 2–4, and day 7 . Control patients had the same assessment at a single time point. RH-PAT (Itamar Medical, Caesarea, Israel) is a non-invasive operator-independent method of assessing endothelial function. Endothelial function is defined by the ability of blood vessels to vasodilate in response to an ischemic stress, which invasive studies have demonstrated to be dependent on endothelial cell NO production . RH-PAT is at least 50% NO-dependent . RH-PAT uses finger probes to measure digital pulse wave amplitude detected by a pressure transducer , and has been validated against the more operator-dependent flow-mediated dilatation method  and with endothelial function in other vascular beds .
Predefined groups for analysis were severe sepsis, non-severe sepsis (meaning sepsis without evidence of organ dysfunction or shock at enrolment), and hospital controls. Continuous parametric variables were compared using Student's t-test or ANOVA while continuous non-parametric variables were compared using Mann-Whitney, Kruskal-Wallis or Wilcoxon tests as appropriate. Correlations were examined using Pearson's or Spearman's tests for parametric and non-parametric data respectively. As SOFA score was highly right-skewed and no transformation gave a normal distribution, Kendall's tau coefficient for partial correlation was used for multivariate analysis involving SOFA . Linear mixed-effects models were used to examine longitudinal correlations. A 2-sided p-value of <0.05 was considered significant. Analyses were performed using Stata version 10.0 (Stata Corp TX, USA) and Prism version 5.01 (GraphPad Software, CA, USA).
The study included 50 patients with severe sepsis, 30 with non-severe sepsis and 40 hospital controls. The three groups did not differ significantly in age or gender (Table 1). Ninety percent of severe sepsis patients and all non-severe sepsis patients were either orally or enterally fed at the time of enrolment; none were receiving parenteral nutrition.
Table 1. Baseline clinical characteristics of participants.doi:10.1371/journal.pone.0021185.t001
IDO activity and sepsis severity
Plasma tryptophan concentrations were significantly reduced in patients with sepsis (p<0.0001, Figure 1 and Table 2). In all sepsis patients, plasma tryptophan was inversely related to SOFA score (r = −0.45, p<0.0001). There was no difference in the baseline plasma tryptophan concentrations among severe sepsis patients who were orally fed (n = 29), enterally fed (n = 16) or who were nil by mouth (n = 5).
Figure 1. Plasma assessment of tryptophan catabolism.
The concentration of plasma tryptophan (Fig. 1A), kynurenine (Fig. 1B) and the KT ratio (Fig. 1C) in 50 severe sepsis patients, 30 non-severe sepsis patients and 40 hospital controls. Fig. 1D shows the KT ratio in severe sepsis patients on admission (n = 50), day 2 (n = 34) and day 7 (n = 16). The KT ratio is determined by dividing the plasma kynurenine concentration (µmol/L) by the plasma tryptophan concentration (µmol/L) and multiplying the quotient by 1000. Horizontal lines represent median values for the group. P value analysis in Figs. 1A–C used a Mann Whitney test, and in Fig. 1D, a paired Wilcoxon test.doi:10.1371/journal.pone.0021185.g001
Table 2. Immunological characteristics of participants (median and interquartile range).doi:10.1371/journal.pone.0021185.t002
Conversely, plasma kynurenine concentrations were elevated in sepsis patients compared to hospital controls (p<0.0001, Figure 1 and Table 2). In all sepsis patients, plasma kynurenine correlated with SOFA score (r = 0.34, p = 0.005). As kynurenine is renally excreted and accumulates in renal failure , , kynurenine concentrations were tested for relationships with renal impairment. Kynurenine concentrations were significantly higher in patients requiring continuous renal replacement therapy (CRRT) (median 4.5 µmol [IQR 4–5.3]) than in patients not receiving CRRT (2.8 µmol [2.1–4.4]; p = 0.03). In all sepsis patients, kynurenine concentration correlated with plasma creatinine (r = 0.41, p = 0.0002). Nevertheless, the association between plasma kynurenine concentration and SOFA score remained significant even after controlling for creatinine (ktau = 0.24, p<0.01).
IDO activity was significantly increased in sepsis patients (median KT ratio 141 [IQR 64–235]) compared to controls (36 [28–52]) (p<0.0001) and in severe sepsis compared to non-severe sepsis (p = 0.0006, Table 2). The baseline KT ratio correlated with APACHE II (rs = 0.51, p<0.0001) and total SOFA scores (rs = 0.54, p<0.0001) in sepsis patients. The KT ratio positively correlated with the hepatic (rs = 0.28, p = 0.01), renal (rs = 0.53, p<0.0001), cardiovascular (rs = 0.42, p<0.0001) and respiratory (rs = 0.36, p = 0.0009) components of the SOFA score but not the coagulation component (rs = 0.13, p = ns).
Of the 80 sepsis patients, 6 died by day 28 of the study. The baseline KT ratio in patients who died (median 270 [IQR 102–431] was not statistically significantly different to those who survived (138 [63–232]; p = 0.2).
In longitudinal analysis of severe sepsis, the KT ratio significantly decreased between day 0 (median 162 [IQR 100–286]) and day 7 (89 [65–139]), p = 0.0006); Figure 1D. Among all sepsis patients, decrease in KT ratio correlated with decrease in SOFA score over time (p<0.0001).
IDO activity and plasma cytokines
Plasma IFN-γ, IL6 and IL10 were all significantly increased in patients with sepsis (Table 2). Plasma concentrations of IL1, IL2, IL4 and tumour necrosis factor-α were not significantly increased in this cohort and were not analysed further. Both IL6 and IL10 positively correlated with SOFA score (rs = 0.55, p<0.0001 and rs = 0.55, p<0.0001 respectively) but there was no association between IFN-γ and SOFA score.
In sepsis patients, the KT ratio correlated with plasma IFN-γ (rs = 0.44, p = 0.0002), IL6 (rs = 0.49, p<0.0001) and IL10 (rs = 0.62, p<0.0001). The associations between KT ratio and IL6 and IL10 remained significant after controlling for SOFA score (ktau = 0.30, p<0.003 and ktau = 0.45, p<0.0001 respectively).
In a univariate mixed effects model, the decrease in KT ratio over time correlated with the decrease in IL6 (p<0.0001) and IL10 (p<0.0001) between day 0 and day 7. In a multivariate model, these relationships remained significant after controlling for change in SOFA score (IL6 p = 0.009; IL10 p = 0.02).
IDO activity and lymphocyte counts
Sepsis patients had increased white blood cell counts (p<0.0001) primarily due to increased circulating neutrophils (p<0.05; Table 2), which proliferate in response to bacterial infections . Conversely, sepsis patients had significantly lower total lymphocyte counts compared with hospital controls (p<0.0001, Table 2). In all sepsis patients the baseline KT ratio was weakly associated with absolute lymphocyte count (rp = 0.26, p = 0.02). In a linear mixed effects model, absolute lymphocyte count increased as the KT ratio decreased over time (p = 0.001). This relationship persisted after controlling for SOFA score (p = 0.008). When all subjects were grouped according to lymphopenia, lymphopenic patients (n = 63) had a median KT ratio of 128 [IQR 63–236], compared with 59 [33–86] in non-lymphopenic patients (n = 57) (p<0.0001).
As IDO activity contributes to T cell apoptosis , we examined the relationship between KT ratio and lymphocyte subsets. Peripheral blood mononuclear cells were analysed from 23 of the 80 sepsis patients whose blood had been processed within 30 minutes of collection. This subset of patients was representative of the cohort in terms of age, gender distribution, total lymphocyte count and KT ratio. In this subset of patients, the KT ratio negatively correlated with absolute numbers of lymphocytes (rp = −0.54, p = 0.007), T cells (rp = −0.53, p = 0.01), CD4+ T cells (rp = −0.50, p = 0.01), CD8+ T cells (rp = −0.49, p = 0.02) and natural killer cells (rp = −0.46, p = 0.03) (Table 2).
IDO activity and endothelial function
In sepsis, the KT ratio at baseline correlated inversely with NO-dependent microvascular reactivity (rs = −0.45, p = 0.001) even after controlling for disease severity (using SOFA score; p = 0.001). In a multivariate mixed effects model controlling for SOFA score, improvement in KT ratio between day 0 and day 7 correlated with improvement in microvascular reactivity (p = 0.001). In all sepsis patients, there was an inverse association between the baseline KT ratio and mean arterial pressure (rs = −0.29, p = 0.009) and diastolic blood pressure (rs = −0.29, p = 0.01) but no association with systolic blood pressure.
IDO activity is increased in sepsis, in proportion to disease severity. IDO-mediated tryptophan catabolism is associated with dysregulated immune responses and impaired microvascular reactivity in sepsis. IFN-γ and IL10 are associated with, and may contribute to, increased IDO activity in sepsis. The independent inverse longitudinal association with total lymphocyte counts suggests a potential role in sepsis-associated lymphopenia. Similarly, the independent inverse association between the KT ratio and microvascular reactivity suggests that IDO activity may also contribute to impaired endothelial function in sepsis. Based on these associations we propose a model of interpretation outlined in Figure 2.
Figure 2. Proposed model of tryptophan catabolism in sepsis.
IDO = Indoleamine 2,3-dioxygenase, IL6 = interleukin-6, IL10 = interleukin-10, IFN-γ = interferon gamma and NO = nitric oxide.doi:10.1371/journal.pone.0021185.g002
Increased expression of IFN-γ , IL6 ,  and IL10  have each been associated with increased tryptophan catabolism by IDO in other disease states. In sepsis patients in our study, IFN-γ concentration correlated with the KT ratio only at baseline, whereas IL6 and IL10 correlated with the KT ratio both at baseline and longitudinally. Our findings agree with the in vitro literature, where IFN-γ induces IDO , . Although under certain conditions, IL-10 has been reported to suppress IDO activity , our findings support the majority of in vitro studies which have shown that IL-10 induces or stabilises IDO , , , . The high IFN-γ associated with early sepsis  may lead to increased IDO activity while high IL10 may sustain or potentially enhance IDO activity  throughout the course of the disease. The role of IL6 in IDO expression is unclear. Orabona et al. suggest that IL6 inhibits IDO activity by increasing murine dendritic cell SOCS3 expression, which drives IDO breakdown . On the other hand, a low tryptophan environment created by IDO activity stabilises IL6 mRNA and increases IL6 responses . Given the conflicting evidence in these and other studies regarding IL6 and IDO, we investigated the relationship between the KT ratio and IL6 in sepsis patients. The strong positive correlation between plasma KT ratio and IL6 concentration is consistent with findings in murine models of sepsis where IDO−/− mice or mice treated with IDO inhibitors have lower plasma IL6 concentrations , .
We report that the high KT ratio in sepsis is associated with a decreased lymphocyte count, independent of disease severity, a finding similar to that found in patients with trauma , human immunodeficiency virus  and cancer . Previous studies in sepsis have associated lymphopenia with disease severity , duration of ICU stay  and mortality  and prevention of lymphocyte apoptosis improves survival in animal models of sepsis , , , . T cells co-cultured with IDO-producing cells have reduced proliferation and increased death , . Both high kynurenine concentrations and low tryptophan concentrations appear to contribute to T cell death. In vivo, kynurenine treatment in mice depletes overall thymocyte counts and, in vitro, thymocytes die of apoptosis when cultured in media with kynurenines . Furthermore, T cells cultured in low tryptophan media have reduced proliferation and increased apoptosis via activated GCN2 kinase , . These in vitro studies suggest a potential mechanism through which increased IDO activity may contribute to lymphopenia and its deleterious consequences in sepsis.
IDO activity regulates vascular tone in sepsis. In this study IDO activity in sepsis patients correlated with diastolic blood pressure but not systolic blood pressure. This is consistent with the recent finding that kynurenine is a vascular relaxation factor . Another important regulator of endothelial function in sepsis is NO. There is significant cross-talk between IDO and NOS, with IDO activity inhibiting both expression and activity of NOS , ,  and vice versa. We found the KT ratio in sepsis is inversely associated with microvascular reactivity as measured by RH-PAT, which is at least 50% dependent on endothelial NO production . Increased IDO activity in sepsis may regulate vascular tone directly, via the vasorelaxing effects of kynurenine, and indirectly, by impairing NO-dependent microvascular reactivity. Increased plasma kynurenine concentrations may further impede endothelial function in sepsis by mediating adhesion of monocytes and neutrophils to the vascular endothelium .
A limitation of this study is that we did not directly measure IDO expression. However, the KT ratio is an established measure of systemic IDO activity ,  with tissue IDO expression and activity directly correlated with plasma KT ratio in multiple human disease states, including celiac disease , hepatitis C  and pre-eclampsia . There are several possible sources of IDO activity in sepsis patients including the endothelium, kidney, liver, lungs and leukocytes , , , , , , although a recent study was unable to detect spontaneous IDO expression in PBMC from sepsis patients . Importantly, the effects of the high KT ratio in sepsis on immune function and endothelial function would be the same whether the high KT ratio was the result of increased IDO activity alone or in combination with decreased feeding and impaired renal excretion of kynurenine. Furthermore, it is unlikely that nutritional deficiency and renal impairment accounted for the differences we found, because controlling for these factors made no difference to our results.
In our study the KT ratio was not significantly associated with mortality. Consistent with the previously reported low mortality from severe sepsis in our ICU , , there were few deaths in our study. This suggests that our study was under-powered to examine the relationship between IDO activity and mortality. However, in a study with higher numbers of deaths, Hattunen and colleagues found a clear association between plasma KT ratio and risk of death in sepsis .
The generation of a low tryptophan environment may be a maladaptive host response to infection. While growth of some bacterial species is inhibited by low tryptophan , most can synthesize tryptophan  and others have specialized tryptophan transport systems . In murine models of sepsis, IDO−/− mice have significantly increased survival compared to wild type mice  and treatment of wild-type mice with IDO inhibitors such as 1-methyl-tryptophan  or ethyl pyruvate also significantly increase survival . The KT ratio is significantly higher in bacteremic patients with a fatal outcome  and we and others have demonstrated that the KT ratio is associated with disease severity in sepsis , , . Together, this evidence supports the hypothesis that increased IDO activity is a deleterious host response in human sepsis. IDO inhibitors are being considered as potential adjunctive cancer treatments  and these treatments may also have therapeutic potential in sepsis.
IDO activity is elevated in sepsis and associated with disease severity, T cell lymphopenia and microvascular dysfunction. Because excessive IDO activity is associated with both immune and endothelial dysfunction, increased tryptophan catabolism may link these two key aspects of sepsis pathophysiology. Modulation of IDO activity warrants investigation as a therapeutic strategy in sepsis.
We thank Kim Piera, Catherine Jones and Barbara MacHunter for laboratory assistance; Jane Thomas, Mark McMillan, Karl Blenk, Antony Van Asche, Steven Tong and Paulene Kittler for RH-PAT measurements and sample collection; Alex Humphrey for database assistance; Joseph McDonnell for statistical advice; David Celermajer for advice on vascular function assessments and contribution to the design of the original study, and the medical and nursing staff of the Royal Darwin Hospital Intensive Care Unit, Division of Medicine and Hospital in the Home.
Conceived and designed the experiments: CJD JSD TW YRM DPS TWY NMA. Performed the experiments: JSD DPS CJD TW YRM. Analyzed the data: CJD JSD TW NMA. Wrote the paper: CJD JSD TW YRM DPS TWY NMA.
- 1. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, et al. (1992) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 101: 1644–1655.
- 2. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, et al. (2001) Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 29: 1303–1310.
- 3. Finfer S, Bellomo R, Lipman J, French C, Dobb G, et al. (2004) Adult-population incidence of severe sepsis in Australian and New Zealand intensive care units. Intensive Care Med 30: 589–596.
- 4. Blanco J, Muriel-Bombin A, Sagredo V, Taboada F, Gandia F, et al. (2008) Incidence, organ dysfunction and mortality in severe sepsis: a Spanish multicentre study. Crit Care 12: R158.
- 5. Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis. N Engl J Med 348: 138–150.
- 6. Aird WC (2003) The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 101: 3765–3777.
- 7. Ellinger P, Abdel Kader MM (1947) Tryptophane as precursor of nicotinamide in mammals. Nature 160: 675.
- 8. Fernstrom JD, Wurtman RJ (1971) Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173: 149–152.
- 9. Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, et al. (2010) Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med 16: 279–285.
- 10. Carlin JM, Borden EC, Sondel PM, Byrne GI (1989) Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes. J Leukoc Biol 45: 29–34.
- 11. Larrea E, Riezu-Boj JI, Gil-Guerrero L, Casares N, Aldabe R, et al. (2007) Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection. J Virol 81: 3662–3666.
- 12. Iwamoto N, Ito H, Ando K, Ishikawa T, Hara A, et al. (2009) Upregulation of indoleamine 2,3-dioxygenase in hepatocyte during acute hepatitis caused by hepatitis B virus-specific cytotoxic T lymphocytes in vivo. Liver Int 29: 277–283.
- 13. Mohib K, Guan Q, Diao H, Du C, Jevnikar AM (2007) Proapoptotic activity of indoleamine 2,3-dioxygenase expressed in renal tubular epithelial cells. Am J Physiol Renal Physiol 293: F801–812.
- 14. Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, et al. (2002) Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297: 1867–1870.
- 15. Fallarino F, Grohmann U, You S, McGrath BC, Cavener DR, et al. (2006) The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor zeta-chain and induce a regulatory phenotype in naive T cells. J Immunol 176: 6752–6761.
- 16. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, et al. (1999) Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med 189: 1363–1372.
- 17. Boasso A, Herbeuval JP, Hardy AW, Anderson SA, Dolan MJ, et al. (2007) HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in plasmacytoid dendritic cells. Blood 109: 3351–3359.
- 18. Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, et al. (2002) T cell apoptosis by tryptophan catabolism. Cell Death Differ 9: 1069–1077.
- 19. Sekkai D, Guittet O, Lemaire G, Tenu JP, Lepoivre M (1997) Inhibition of nitric oxide synthase expression and activity in macrophages by 3-hydroxyanthranilic acid, a tryptophan metabolite. Arch Biochem Biophys 340: 117–123.
- 20. Chiarugi A, Rovida E, Dello Sbarba P, Moroni F (2003) Tryptophan availability selectively limits NO-synthase induction in macrophages. J Leukoc Biol 73: 172–177.
- 21. Samelson-Jones BJ, Yeh SR (2006) Interactions between nitric oxide and indoleamine 2,3-dioxygenase. Biochemistry 45: 8527–8538.
- 22. Luiking YC, Poeze M, Ramsay G, Deutz NE (2009) Reduced citrulline production in sepsis is related to diminished de novo arginine and nitric oxide production. Am J Clin Nutr 89: 142–152.
- 23. Kao CC, Bandi V, Guntupalli KK, Wu M, Castillo L, et al. (2009) Arginine, citrulline and nitric oxide metabolism in sepsis. Clin Sci (Lond) 117: 23–30.
- 24. Villalpando S, Gopal J, Balasubramanyam A, Bandi VP, Guntupalli K, et al. (2006) In vivo arginine production and intravascular nitric oxide synthesis in hypotensive sepsis. Am J Clin Nutr 84: 197–203.
- 25. Davis JS, Yeo TW, Thomas JH, McMillan M, Darcy CJ, et al. (2009) Sepsis-associated microvascular dysfunction measured by peripheral arterial tonometry: an observational study. Crit Care 13: R155.
- 26. Vaudo G, Marchesi S, Siepi D, Brozzetti M, Lombardini R, et al. (2008) Human endothelial impairment in sepsis. Atherosclerosis 197: 747–752.
- 27. Pawlak K, Domaniewski T, Mysliwiec M, Pawlak D (2009) Kynurenines and oxidative status are independently associated with thrombomodulin and von Willebrand factor levels in patients with end-stage renal disease. Thromb Res 124: 452–457.
- 28. Huengsberg M, Winer JB, Gompels M, Round R, Ross J, et al. (1998) Serum kynurenine-to-tryptophan ratio increases with progressive disease in HIV-infected patients. Clin Chem 44: 858–862.
- 29. Widner B, Sepp N, Kowald E, Ortner U, Wirleitner B, et al. (2000) Enhanced tryptophan degradation in systemic lupus erythematosus. Immunobiology 201: 621–630.
- 30. Huang A, Fuchs D, Widner B, Glover C, Henderson DC, et al. (2002) Serum tryptophan decrease correlates with immune activation and impaired quality of life in colorectal cancer. Br J Cancer 86: 1691–1696.
- 31. Huttunen R, Syrjanen J, Aittoniemi J, Oja SS, Raitala A, et al. (2009) High activity of indoleamine 2,3 dioxygenase enzyme predicts disease severity and case fatality in bacteremic patients. Shock.
- 32. Stephens DP, Thomas JH, Higgins A, Bailey M, Anstey NM, et al. (2008) Randomized, double-blind, placebo-controlled trial of granulocyte colony-stimulating factor in patients with septic shock. Crit Care Med 36: 448–454.
- 33. Vincent JL, de Mendonca A, Cantraine F, Moreno R, Takala J, et al. (1998) Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Working group on “sepsis-related problems” of the European Society of Intensive Care Medicine. Crit Care Med 26: 1793–1800.
- 34. Hotchkiss RS, Swanson PE, Freeman BD, Tinsley KW, Cobb JP, et al. (1999) Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 27: 1230–1251.
- 35. van Wandelen C, Cohen SA (1997) Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquionolyl-N-hydroxysuccinyl carbamate-derivatized amino acid mixtures. J Chromatogr A 763: 11–22.
- 36. Zangerle R, Widner B, Quirchmair G, Neurauter G, Sarcletti M, et al. (2002) Effective antiretroviral therapy reduces degradation of tryptophan in patients with HIV-1 infection. Clin Immunol 104: 242–247.
- 37. Pellegrin K, Neurauter G, Wirleitner B, Fleming AW, Peterson VM, et al. (2005) Enhanced enzymatic degradation of tryptophan by indoleamine 2,3-dioxygenase contributes to the tryptophan-deficient state seen after major trauma. Shock 23: 209–215.
- 38. Deanfield JE, Halcox JP, Rabelink TJ (2007) Endothelial function and dysfunction: testing and clinical relevance. Circulation 115: 1285–1295.
- 39. Kuvin JT, Mammen A, Mooney P, Alsheikh-Ali AA, Karas RH (2007) Assessment of peripheral vascular endothelial function in the ambulatory setting. Vasc Med 12: 13–16.
- 40. Celermajer DS (2008) Reliable endothelial function testing: at our fingertips? Circulation 117: 2428–2430.
- 41. Kuvin JT, Patel AR, Sliney KA, Pandian NG, Sheffy J, et al. (2003) Assessment of peripheral vascular endothelial function with finger arterial pulse wave amplitude. Am Heart J 146: 168–174.
- 42. Bonetti PO, Pumper GM, Higano ST, Holmes DR Jr, Kuvin JT, et al. (2004) Noninvasive identification of patients with early coronary atherosclerosis by assessment of digital reactive hyperemia. J Am Coll Cardiol 44: 2137–2141.
- 43. Gibbons J, Chakraborti S (2003) Nonparametric Statistical Inference. Marcel Dekker.
- 44. Pawlak D, Tankiewicz A, Matys T, Buczko W (2003) Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure. J Physiol Pharmacol 54: 175–189.
- 45. Schefold JC, Zeden JP, Fotopoulou C, von Haehling S, Pschowski R, et al. (2009) Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease: a possible link between chronic inflammation and uraemic symptoms. Nephrol Dial Transplant 24: 1901–1908.
- 46. Nelson S (1994) Role of granulocyte colony-stimulating factor in the immune response to acute bacterial infection in the nonneutropenic host: an overview. Clin Infect Dis 18: Suppl 2S197–204.
- 47. Yoshida R, Imanishi J, Oku T, Kishida T, Hayaishi O (1981) Induction of pulmonary indoleamine 2,3-dioxygenase by interferon. Proc Natl Acad Sci U S A 78: 129–132.
- 48. Maes M, Meltzer HY, Scharpe S, Bosmans E, Suy E, et al. (1993) Relationships between lower plasma L-tryptophan levels and immune-inflammatory variables in depression. Psychiatry Res 49: 151–165.
- 49. Bonaccorso S, Lin A, Song C, Verkerk R, Kenis G, et al. (1998) Serotonin-immune interactions in elderly volunteers and in patients with Alzheimer's disease (DAT): lower plasma tryptophan availability to the brain in the elderly and increased serum interleukin-6 in DAT. Aging (Milano) 10: 316–323.
- 50. MacKenzie CR, Gonzalez RG, Kniep E, Roch S, Daubener W (1999) Cytokine mediated regulation of interferon-gamma-induced IDO activation. Adv Exp Med Biol 467: 533–539.
- 51. van der Sluijs KF, Nijhuis M, Levels JH, Florquin S, Mellor AL, et al. (2006) Influenza-induced expression of indoleamine 2,3-dioxygenase enhances interleukin-10 production and bacterial outgrowth during secondary pneumococcal pneumonia. J Infect Dis 193: 214–222.
- 52. Maneechotesuwan K, Supawita S, Kasetsinsombat K, Wongkajornsilp A, Barnes PJ (2008) Sputum indoleamine-2, 3-dioxygenase activity is increased in asthmatic airways by using inhaled corticosteroids. J Allergy Clin Immunol 121: 43–50.
- 53. Yanagawa Y, Iwabuchi K, Onoe K (2009) Co-operative action of interleukin-10 and interferon-gamma to regulate dendritic cell functions. Immunology 127: 345–353.
- 54. Hunsicker A, Kullich W, Weissenhofer W, Lorenz D, Petermann J, et al. (1997) Correlations between endotoxin, interferon-gamma, biopterin and serum phospholipase A2-activities during lethal gram negative sepsis in rats. Eur J Surg 163: 379–385.
- 55. Orabona C, Pallotta MT, Volpi C, Fallarino F, Vacca C, et al. (2008) SOCS3 drives proteasomal degradation of indoleamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Proc Natl Acad Sci U S A 105: 20828–20833.
- 56. van Wissen M, Snoek M, Smids B, Jansen HM, Lutter R (2002) IFN-gamma amplifies IL-6 and IL-8 responses by airway epithelial-like cells via indoleamine 2,3-dioxygenase. J Immunol 169: 7039–7044.
- 57. Ulloa L, Ochani M, Yang H, Tanovic M, Halperin D, et al. (2002) Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci U S A 99: 12351–12356.
- 58. Jung ID, Lee MG, Chang JH, Lee JS, Jeong YI, et al. (2009) Blockade of indoleamine 2,3-dioxygenase protects mice against lipopolysaccharide-induced endotoxin shock. J Immunol 182: 3146–3154.
- 59. Ino K, Yamamoto E, Shibata K, Kajiyama H, Yoshida N, et al. (2008) Inverse correlation between tumoral indoleamine 2,3-dioxygenase expression and tumor-infiltrating lymphocytes in endometrial cancer: its association with disease progression and survival. Clin Cancer Res 14: 2310–2317.
- 60. Le Tulzo Y, Pangault C, Gacouin A, Guilloux V, Tribut O, et al. (2002) Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome. Shock 18: 487–494.
- 61. Felmet KA, Hall MW, Clark RS, Jaffe R, Carcillo JA (2005) Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure. J Immunol 174: 3765–3772.
- 62. Hotchkiss RS, Tinsley KW, Swanson PE, Chang KC, Cobb JP, et al. (1999) Prevention of lymphocyte cell death in sepsis improves survival in mice. Proc Natl Acad Sci U S A 96: 14541–14546.
- 63. Hotchkiss RS, Chang KC, Swanson PE, Tinsley KW, Hui JJ, et al. (2000) Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat Immunol 1: 496–501.
- 64. Bommhardt U, Chang KC, Swanson PE, Wagner TH, Tinsley KW, et al. (2004) Akt decreases lymphocyte apoptosis and improves survival in sepsis. J Immunol 172: 7583–7591.
- 65. Schwulst SJ, Muenzer JT, Peck-Palmer OM, Chang KC, Davis CG, et al. (2008) Bim siRNA decreases lymphocyte apoptosis and improves survival in sepsis. Shock 30: 127–134.
- 66. Fallarino F, Vacca C, Orabona C, Belladonna ML, Bianchi R, et al. (2002) Functional expression of indoleamine 2,3-dioxygenase by murine CD8 alpha(+) dendritic cells. Int Immunol 14: 65–68.
- 67. Odemuyiwa SO, Ghahary A, Li Y, Puttagunta L, Lee JE, et al. (2004) Cutting edge: human eosinophils regulate T cell subset selection through indoleamine 2,3-dioxygenase. J Immunol 173: 5909–5913.
- 68. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, et al. (2002) Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 107: 452–460.
- 69. Forouzandeh F, Jalili RB, Germain M, Duronio V, Ghahary A (2008) Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts. Wound Repair Regen 16: 379–387.
- 70. Nohria A, Gerhard-Herman M, Creager MA, Hurley S, Mitra D, et al. (2006) Role of nitric oxide in the regulation of digital pulse volume amplitude in humans. J Appl Physiol 101: 545–548.
- 71. Barth MC, Ahluwalia N, Anderson TJ, Hardy GJ, Sinha S, et al. (2009) Kynurenic acid triggers firm arrest of leukocytes to vascular endothelium under flow conditions. J Biol Chem.
- 72. Suzuki Y, Suda T, Furuhashi K, Suzuki M, Fujie M, et al. (2010) Increased serum kynurenine/tryptophan ratio correlates with disease progression in lung cancer. Lung Cancer 67: 361–365.
- 73. Torres MI, Lopez-Casado MA, Lorite P, Rios A (2007) Tryptophan metabolism and indoleamine 2,3-dioxygenase expression in coeliac disease. Clin Exp Immunol 148: 419–424.
- 74. Kudo Y, Boyd CA, Sargent IL, Redman CW (2003) Decreased tryptophan catabolism by placental indoleamine 2,3-dioxygenase in preeclampsia. Am J Obstet Gynecol 188: 719–726.
- 75. Tattevin P, Monnier D, Tribut O, Dulong J, Bescher N, et al. (2010) Enhanced indoleamine 2,3-dioxygenase activity in patients with severe sepsis and septic shock. J Infect Dis 201: 956–966.
- 76. Davis JS, Cheng AC, McMillan M, Anstey NM (2011) Sepsis in the tropical Northern Territory of Australia: high disease burden with disproportionate impact on Indigenous Australians. Med J Aust. In press.
- 77. MacKenzie CR, Hadding U, Daubener W (1998) Interferon-gamma-induced activation of indoleamine 2,3-dioxygenase in cord blood monocyte-derived macrophages inhibits the growth of group B streptococci. J Infect Dis 178: 875–878.
- 78. Merino E, Jensen RA, Yanofsky C (2008) Evolution of bacterial trp operons and their regulation. Curr Opin Microbiol 11: 78–86.
- 79. Yanofsky C, Horn V, Gollnick P (1991) Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli. J Bacteriol 173: 6009–6017.
- 80. Schefold JC, Zeden JP, Pschowski R, Hammoud B, Fotopoulou C, et al. (2010) Treatment with granulocyte-macrophage colony-stimulating factor is associated with reduced indoleamine 2,3-dioxygenase activity and kynurenine pathway catabolites in patients with severe sepsis and septic shock. Scand J Infect Dis 42: 164–171.
- 81. Lob S, Konigsrainer A, Rammensee HG, Opelz G, Terness P (2009) Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees? Nat Rev Cancer 9: 445–452.