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VG-CARE – Vietnamese–German Centre for Medical Research, VG-CARE, Hanoi, Viet NamInstitute of Tropical Medicine, University of Tübingen, Tübingen, Germany
1 Dietmar Fuchs and Thirumalaisamy P. Velavan are joint senior authors.
Affiliations
VG-CARE – Vietnamese–German Centre for Medical Research, VG-CARE, Hanoi, Viet NamInstitute of Tropical Medicine, University of Tübingen, Tübingen, GermanyFaculty of Medicine, Duy Tan University, Da Nang, Viet Nam
Neopterin levels and Kyn/Trp ratios were significantly increased in dengue virus (DENV) patients and subsequently decreased after recovery.
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Neopterin concentrations strongly correlated with Kyn/Trp ratios.
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Neopterin levels and Kyn/Trp ratios could be used as indicators of the response to supportive care in DENV infection.
Abstract
Objectives
During dengue fever, a pronounced gamma-interferon immune response produces neopterin and promotes tryptophan degradation by the enzyme indoleamine-2,3-dioxygenase 1 (IDO-1). Activated IDO-1 is indicated by an increased kynurenine to tryptophan ratio (Kyn/Trp) in patients.
Methods
Plasma levels of neopterin, kynurenine, and tryptophan were measured in 72 hospitalized dengue virus (DENV) patients and 100 healthy individuals. Plasma levels of neopterin, kynurenine, and tryptophan were also measured prospectively in a second cohort of 13 DENV patients; on the day of hospitalization, on day 2–3 at discharge, and 7–10 days after discharge. DENV RNA positivity was determined by qualitative and quantitative methodologies.
Results
DENV RNA-positive patients presented significantly higher levels of neopterin (mean 36.5 nmol/l) and Kyn/Trp ratios (mean 102 μmol/mmol) compared to DENV RNA-negative individuals. A significant correlation between neopterin levels and Kyn/Trp ratios was observed in both DENV RNA-positive (Spearman’s rho = 0.37, p < 0.01) and DENV RNA-negative (Spearman’s rho = 0.89, p < 0.001) patients. Kyn/Trp ratios were negatively correlated with platelet counts (Spearman’s rho = −0.43, p < 0.01) and positively correlated with liver enzymes: AST (Spearman’s rho = 0.68, p < 0.01) and ALT (Spearman’s rho = 0.51, p < 0.05). In addition, the follow-up data presented a significant decrease in neopterin levels and Kyn/Trp ratios within 10 days after hospital entry.
Conclusions
Neopterin levels and Kyn/Trp ratios were significantly increased in DENV patients and subsequently decreased after recovery.
Dengue caused by dengue virus (DENV) is an acute viral disease endemic in the tropics, with an increasing incidence greater than 30-fold since 1960. Currently, 96 million clinically manifested DENV infections and 390 million DENV infections are reported each year (
). Four closely related DENV serotypes have been identified, based on the antigenicity of the non-structural envelope protein (NS1): DENV-1, DENV-2, DENV-3, and DENV-4 (
). The clinical presentation of an acute DENV infection varies from a mild febrile illness that lasts approximately 4–7 days to life-threatening severe dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS), which includes plasma leakage, severe bleeding, and organ complications (
Langerhans cells, a type of dendritic cell (DC), present the viral antigens and activate monocytes and macrophages. However, it is believed that the monocytes and macrophages are actually being infected by the virus, thus spreading the virus in the lymphatic system, resulting in viral dissemination. During this process, DCs and macrophages release a wide range of cytokines and chemokines (
). Various studies have reported the signature cytokine profiles during dengue fever (DF) and DHF/DSS, with decreased lymphocytes and increased levels of urinary histamine (
). The significant correlation between serotonin levels and acute thrombocytopenia indicates a strong dependence of platelet function on serotonin availability (
During DENV infection, the Th1-type immune responses are activated, in which the infected cells secrete interferon-gamma (IFN-γ) to protect uninfected cells from DENV and modulate the viral replication process. IFN-γ also induces the release of neopterin and the breakdown of essential amino acid tryptophan (Trp) by the enzyme indoleamine-2,3-dioxygenase 1 (IDO-1). IDO-1 degrades the essential amino acid Trp to form kynurenine (Kyn) (
). The correlation of IDO-1 activity with neopterin levels reliably estimates the IFN-γ-induced immune activation. IDO-1 activity and Kyn levels result in the maturation of functional regulatory T-cells (Tregs) and natural killer (NK) cells, in particular the differentiation of Th17 cells to Tregs. Although CD4+ CD25+ FoxP3+ T-cells are expanded during acute dengue, there is no correlation with the suppression of anti-dengue T-cell responses, viremia, and pathogenesis (
). The depletion of amino acid Trp by immune cells is an indicator of depleting growth of pathogens and tumour cells. To date, only one study has described depleted Trp and serotonin concentrations with increased Kyn/Trp and a trend towards higher IDO-1 activity in primary rather than secondary infection during the febrile phase of dengue infection (
The anti-proliferative effect of IFN-γ induces neopterin, which is synthesized primarily by the macrophages and DCs, and is well established for cellular immune system activation (
). Neopterin concentrations in body fluids are useful in monitoring cellular immune activation (Th1-type) during infections, autoimmune diseases, and malignancies. Increased neopterin concentrations are observed prior to detectable viral antigen-specific IgM antibodies in HIV type 1 (HIV-1), cytomegalovirus (CMV), and rubella infections (
). In the present study, the neopterin, Trp, and Kyn levels were investigated in plasma samples of dengue patients and healthy control subjects, as well as their association with clinical data.
Materials and methods
Patients
Eighty five dengue patients (n = 85) were recruited in accordance with World Health Organization (WHO) guidelines (
), during the seasonal outbreak November 2016 to February 2017. The first cohort, 72 febrile patients (38 male, 34 female; mean age 33.2 ± 14.9 years) were evaluated on the day of hospital entry and on the day of discharge (2–3 days after supportive care treatment). The second cohort of 13 patients (five male, eight female; mean age 31 ± 10.3 years) was followed up. Blood samples of these 13 patients were collected at three time intervals: on the day of hospitalization (within 6 days of their first reported fever), on day 2–3 of hospitalization, and on day 7–10 after discharge. On the day of admission, all patients were screened for NS1 and IgG/IgM by rapid combi diagnostic test (SD Bioline Inc., Seoul, South Korea). Based on NS1 or IgM positivity and/or the severity of their signs and symptoms, the patients were provided with supportive treatment. The study design excluded patients who had any underlying chronic disease, including chronic viral infections, diabetes, and autoimmune diseases. The EDTA plasma of DENV patients was stored at −80 °C until further use. As control subjects, healthy individuals who were blood donors were recruited from the Central Institute of Blood Transfusion and Immunology of the University Clinics Innsbruck. Their demographic data and other clinical parameters have been described in a previously published study (
DENV, dengue virus; AST, aspartate aminotransferase; ALT, alanine aminotransferase; pos, positive; neg, negative; NS, not significant; ND, not determined. Values given are the median and range.
a p-Values were calculated by Mann–Whitney U-test.
Written informed consent was obtained from all study participants (parents/guardians in the case of children) prior to recruitment. The study was approved by the Institutional Review Board of Vietnam Military Medical University, Hanoi, Vietnam (103MCH/RES/DENV-GER_V-D1-2016).
DENV RNA and DENV-specific antibodies
Viral RNA testing was performed on cohort 1 (n = 72) by automated RT-PCR Cobas TaqMan AmpliPrep system (Roche, Penzberg, Germany). For cohort 2 (n = 13), this was done by manual RNA isolation and ModularDx Dengue Kit (58-0700-96) with LightMix EAV extraction control (66-0909-96) and DENV1–4 serotyping (Dengue Typing 40-0700-2) (TIB Molbiol, Berlin, Germany) on a Roche Light Cycler 480 system. DENV-specific IgM and IgG antibodies were determined by ELISA (Serion ELISA classic, dengue virus IgM from Virion; Serion GmbH, Würzburg, Germany), following the manufacturer’s instructions. The measurement ranges for quantification were 5–200 U/ml for the IgM test and 5–600 U/ml for the IgG test, with a cut-off of >15 U/l.
Neopterin, tryptophan, and kynurenine levels
Neopterin levels were determined by ELISA (BRAHMS GmbH, Hennigsdorf, Germany), following the manufacturer’s instructions. The sensitivity of the test was 2 nmol/l neopterin. Trp and Kyn concentrations were determined by reverse-phase high-performance liquid chromatography (HPLC) method, as described previously (
), using a Varian ProStar HPLC system with a LiChrosorb C18 column (3 μm particle size; Merck, Darmstadt, Germany) and 15 mmol/l acetic acid–sodium acetate solution (pH 4.0) as the mobile phase. Kyn and internal standard 3-nitro-l-tyrosine concentrations were determined by a UV-spectrometric detector (SPD-6A, Shimadzu) at a wavelength of 360 nm. Trp was detected by natural fluorescence at an excitation wavelength of 286 nm and an emission wavelength of 366 nm using a fluorescence detector (Model 360, Varian ProStar). The Kyn/Trp ratio was calculated and presented as μmol/mmol.
Statistical analysis
Neopterin and amino acid levels are presented as the mean ± standard deviation values and as the median with range. The non-parametric Mann–Whitney U-test was performed to compare quantitative variables between groups. Correlation analyses were conducted with the Spearman rank correlation coefficient. The statistical analyses were performed using IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA). Significance was set at a value of p < 0.05.
Results
Dengue patients
In cohort 1, 56 of the 72 patients tested were found to be DENV RNA-positive on hospitalization. Of the 56 patients positive for DENV RNA, 45 were positive by IgG ELISA, 42 were positive by IgM ELISA, and 20 patients were positive for NS1. The remaining 16 of 72 patients (negative for DENV RNA) were negative by the NS1 rapid test. Among the 16 patients who were negative for DENV RNA and NS1, four who showed a clinical presentation of DF according to WHO guidelines were negative for anti-DENV IgM and IgG by ELISA. The remaining 12 patients who were negative for DENV RNA but had a clinical presentation were positive either for anti-DENV IgM or anti-DENV IgG by ELISA. The median age of the 56 patients who were positive for DENV (29.5 years) did not differ significantly from the median age of the 16 patients who were negative for DENV (37 years). Also, there was no significant difference in sex ratio between the patients who were positive and negative for DENV (Table 1).
In the second cohort with a prospective follow-up, 12 of the 13 patients were confirmed for DENV RNA: 10 were infected with the DENV-1 serotype and two with the DENV-2 serotype. One of the 13 patients remained RNA-negative and was also negative by IgM and IgG ELISA on the day of hospital entry (7 U/l and 9 U/l, respectively), but had a positive NS1 rapid test result. This individual had undergone seroconversion with IgM 16–63 U/l and IgG 273 U/l levels on day 10 after hospital discharge (Table 1).
Serum concentrations of neopterin and Kyn/Trp ratios
Serum neopterin concentrations were >10 nmol/l in all tested patients, with a mean concentration of 33.8 ± 13.3 nmol/l (range 11.2–70.9 nmol/l) on the day of hospital admission. The neopterin concentrations in DENV RNA-positive individuals (36.5 ± 11.7 nmol/l) were observed to be higher than those in DENV RNA-negative patients (p = 0.003). DENV RNA-positive patients presented lower Trp concentrations (39.3 ± 12.7 μmol/l) than DENV RNA-negative patients (52.2 ± 14.3 μmol/l) (p = 0.001). DENV RNA-positive individuals presented higher Kyn concentrations (3.57 ± 1.4 μmol/l) than DENV-negative individuals (3.34 ± 1.2 μmol/l) (p = 0.95) (Figure 1). DENV RNA-positive patients had significantly higher Kyn/Trp ratios than DENV RNA-negative patients (p = 0.026) (Figure 1).
Figure 1Neopterin concentrations and kynurenine to tryptophan (Kyn/Trp) ratio in the study groups.
The concentrations of neopterin (A), tryptophan (B), and kynurenine (C) were measured in the plasma of the study patients. The kynurenine to tryptophan (Kyn/Trp) ratios were also calculated and compared between the groups (D). DENV pos: patients positive for dengue virus infection by PCR; DENV neg: patients negative for dengue virus infection by PCR; Controls: healthy individuals. p-Values were calculated by Mann–Whitney U-test. Dotted lines represent the upper limit of the reference range.
Correlation of neopterin levels and Kyn/Trp ratios
In patients positive for DENV RNA, neopterin levels were positively correlated with Kyn concentrations (Spearman’s rho = 0.53; p < 0.0001) (Figure 2A) and with Kyn/Trp ratios (Spearman’s rho = 0.37; p = 0.005) (Figure 2B). Importantly, platelet counts were positively correlated with Trp concentrations (Spearman’s rho = 0.26; p = 0.06) (Figure 2C) and, as expected, were negatively correlated with Kyn/Trp ratios (Spearman’s rho = −0.43; p = 0.001) (Figure 2D). In the group of patients negative for DENV RNA, neopterin concentrations were negatively correlated with Trp concentrations (Spearman’s rho = −0.62; p = 0.011) (Figure 3A) and positively correlated with Kyn concentrations (Spearman’s rho = 0.46; p = 0.077) (Figure 3B) and with Kyn/Trp ratios (Spearman’s rho = 0.85; p < 0.0001) (Figure 3C). The Kyn/Trp ratios were positively correlated with aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Spearman’s rho = 0.68 and 0.51; p = 0.005 and 0.046, respectively) (Figure 3D, E). The correlation between neopterin levels and Kyn/Trp ratios in DENV RNA-negative individuals was stronger than that in DENV RNA-positive patients (Figure 2B and Figure 3C).
Figure 2Correlation analyses in DENV RNA-positive patients.
Correlation between parameters was analysed using the Spearman rank correlation coefficients in DENV RNA-positive patients. Spearman’s rho and p-values were calculated and are presented.
Correlation between parameters was analysed using the Spearman rank correlation coefficients in DENV RNA-negative patients. Spearman’s rho and p-values were calculated and are presented.
Decrease in neopterin levels and Kyn/Trp ratio in follow-up patients
In the longitudinal follow-up of patients, the neopterin concentrations were high during the admission period (60.1 ± 23.4 nmol/l; range 11.1–98.0 nmol/l) and eventually decreased on the day of hospital discharge (57.2 ± 17.0 nmol/l; range 31.8–90.0 nmol/l). In the post follow-up period after 7–10 days, the DENV-infected individuals presented significantly low neopterin concentrations (13.7 ± 5.2 nmol/l; range 4.7–21.1 nmol/l) (p < 0.0001) (Figure 4A). The Trp concentrations were low on admission (43.1 ± 18.8 μmol/l; range 14.5–78.0) and had increased significantly on the day of hospital discharge (60.7 ± 16.3 μmol/l; range 28.7–79.0 μmol/l) and also at 7–10 days after hospital entry (71.4 ± 10.7 μmol/l; range 50.9– 90.8 μmol/l) (p = 0.026 and p < 0.0001, respectively) (Figure 4B). The Kyn concentrations were increased on the day of hospital discharge (5.6 ± 1.5 μmol/l; range 3.0–8.6 μmol/l) compared to those at hospital entry (4.3 ± 2.2 μmol/l; range 2–10.5 μmol/l), but decreased steeply at 7–10 days after hospital entry (2.6 ± 0.8 μmol/l; range 2.0–4.4 μmol/l) (p < 0.05) (Figure 4C). The Kyn/Trp ratios were also compared among the three time points. The mean Kyn/Trp ratio in DF patients was 109.6 ± 43.0 μmol/mmol (range 52.9–190.6 μmol/mmol) at the time point of hospital entry and declined slightly until the date of the hospital discharge (99.4 ± 39.9 μmol/mmol, range 41.8–180.0 μmol/mmol). At 7–10 days after hospital entry, the mean Kyn/Trp ratio had decreased to 37.4 ± 14.0 μmol/mmol (range 22.8–68.9 μmol/mmol) (p < 0.0001) (Figure 4D).
Figure 4Neopterin concentrations and kynurenine to tryptophan (Kyn/Trp) ratio in the follow-up patients.
The concentrations of neopterin, kynurenine, and tryptophan were measured in serum samples of the follow-up patients, obtained at hospital entry, hospital discharge on day 2–3, and 7–10 days after hospital discharge during follow-up. The kynurenine to tryptophan (Kyn/Trp) ratios were also calculated. p-Values were calculated by Mann–Whitney U-test and are presented in the figures. Dotted lines represent the upper limit of the reference range.
An elevated Kyn/Trp ratio is a direct indicator of activated IDO-1 activity, because both biochemical events, neopterin production and IDO-1 activation, are induced by pro-inflammatory stimuli like IFN-γ (
). The present study showed high Kyn/Trp ratios in patients with DENV infection that strongly correlated with neopterin concentrations, underscoring the evidence of IFN-γ-induced IDO-1 activity in DENV infection. The Kyn/Trp ratio in DENV-infected patients was significantly increased. The present study findings suggest that although DENV evades the host interferon-beta (IFN-β) and IFN-γ downstream signalling via the NS4B, which inhibits both the IFN-β and IFN-γ signal transducer and activator (STAT-1), viral infection exerts no decrease in upstream IFN-γ signalling via induction of GTP-cyclohydrolase I (neopterin) and IDO-1 (Kyn/Trp). As IDO-1 catalyses the oxidation of Trp to N-formyl-kynurenine in the case of infection, significantly lower Trp concentrations can be observed in DENV-infected patients compared to healthy individuals. In the case of an infection, depletion of the essential amino acid Trp could lead to a decreased production of proteins, which would also reduce the level of virus replication and T-cell proliferation (
). In acute inflammation, early mechanisms for suppression of the immune reaction are necessary, as an overreacting immune system could severely damage cells and tissue (
). Nevertheless, further studies are required to investigate the role of IDO-1 activity in DENV infection, especially when a high and steep increase in Kyn/Trp levels is observed in DENV-infected patients.
The present study outlines Trp degradation and neopterin production in patients who were treated in hospital with an indicated DENV infection. Compared to earlier studies showing high neopterin in patients with DENV infection (
), neopterin concentrations in the present study confirmed these findings. As higher neopterin concentrations in DF patients were shown to be associated with longer fever duration and the severity of inflammation (
), our results indicate a strong correlation of neopterin and Kyn/Trp ratio over a period of nearly 10 days, starting from the time of first diagnosis at the hospital until convalescence. Neopterin is produced by activated monocytes, macrophages, and DCs upon stimulation by IFN-γ and has been a useful marker for cellular immune activation (
) and for monitoring infectious disease activity during treatment. Elevated neopterin levels have been associated with various infectious diseases including viral infections and cancers (
). Although, neopterin may not be a specific marker to differentiate infections, neopterin may still be useful as a non-specific marker of activated cell-mediated immunity and a potential indicator of the treatment response in DENV patients.
Due to the fact that only 56 of the 72 patients classified on clinical grounds were confirmed to be DENV-positive by PCR, and 15 of the remaining 16 patients testing DENV RNA-negative showed either positive IgM, IgG, and/or NS1 serology, we conclude that the 16 DENV RNA-negative patients were sampled after the viremic phase when viral RNA in their plasma was no longer detectable. Such a conclusion would agree with earlier findings indicating elevated neopterin in various viral infections but even higher neopterin concentrations, mostly >30 nmol/l, in cases of DENV infection (
). The present study of DF patients under hospital care supports the view that elevated IDO-1 activity is involved in the decline of Trp. Notably, IDO-1 also utilizes serotonin as a substrate (
), which could directly contribute to the lower serotonin levels in DENV-infected patients. In addition, the lower Trp levels in patients may diminish the production rate of serotonin out of the essential amino acids.
As serotonin is associated with platelet function and enhances platelet aggregation and interaction with other cell types, low serotonin levels could explain the heightened possibility of plasma leakage (haemorrhagic form) in DHF patients (
). As Trp is a precursor in serotonin synthesis, a rapid increase in Trp degradation could contribute to serotonin deficiency in the blood, therefore reduced Trp availability could play a key role in maintaining the integrity of the vascular system and further developing the haemorrhagic form of DENV infection. Decreased Trp concentrations together with the decline in serotonin concentrations may impair platelet function (
High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever.
). In addition, decreased Trp levels in the blood will limit the transport of the amino acid across the blood–brain barrier and diminish serotonin availability for the brain, which might manifest in altered mood and depressive symptoms (
). Therefore, early assessment of Trp degradation through the measurement of Trp concentrations in the blood of patients could be used to provide proper medical treatment to patients with an increased likelihood of developing DHF.
In conclusion, this study showed that neopterin levels and Kyn/Trp ratios were significantly increased in DENV patients and subsequently decreased after recovery. Neopterin concentrations were also strongly correlated with Kyn/Trp ratios. These results indicate that neopterin levels and Kyn/Trp ratios could be utilized as indicators of the response to supportive care of DENV infection. Nevertheless, further studies are warranted to substantiate these findings and to define the optimal cut-off values for patients with DENV of varying clinical severity involving haemorrhagic and non-haemorrhagic fever during the viremic phase of all four serotypes, at follow-up time intervals after the onset of febrile illness, and during recovery from infection.
Author contributions
SG performed experiments; SDL designed the study and wrote the manuscript; NLT, TTN, NMN, HVH, NTS, DTA, HTT, TVT, and DQ were involved in the recruitment of patients; HVT and NXH analysed the data; LHS and SRP contributed to the study design and analysis; JM and DF contributed to materials and reagents for the study; TPV designed the study, analysed the data, and wrote the manuscript.
Funding
The study was funded by an internal grant from the Vietnamese–German Centre for Medical Research . This study was supported by DAAD-PAGEL ( 57445019 ) for a student fellowship.
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgements
We thank all of the patients and the healthy individuals for their participation. We thank Dr P. Pranav and Dr O. Landt, TIB Molbiol for the RNA extractions, RT-PCR amplification, and determination of the dengue virus serotype (1–4) for the 13 dengue patients.
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