Assessment of neopterin and indoleamine 2,3‐dioxygenase activity in patients with seasonal influenza: A pilot study

Abstract Background Seasonal influenza is an important cause of morbidity and mortality worldwide. Immune activation after stimulation with interferon‐gamma leads to increased production of neopterin but also results in increased tryptophan catabolism through indoleamine 2,3‐dioxygenase (IDO). Our pilot study determined neopterin serum levels and IDO activity in patients with influenza infection and investigated whether neopterin is linked to clinical outcome parameters (mortality ≤30 days, acute cardiac events (ACE) length of hospitalization, ICU admission). Methods Neopterin concentrations were analyzed in serum samples of 40 patients with a confirmed diagnosis of influenza infection and in‐hospital treatment for >24 hours. Data were compared to values of 100 healthy blood donors and 48 age‐matched pneumonia patients. In a subgroup of 14 patients, tryptophan and kynurenine concentrations, as well as kynurenine‐to‐tryptophan ratio, were analyzed. Results In all influenza patients, neopterin concentrations were increased and significantly higher compared to those determined in patients with pneumonia and healthy controls. Positive correlations between the duration of hospitalization and neopterin were found. Significantly higher levels of kynurenine, kynurenine‐to‐tryptophan ratio, and lower levels of tryptophan were seen in influenza patients compared to healthy controls. Conclusions Neopterin seems to be related to the course of the disease and could be a valuable biomarker to identify patients at an elevated risk of a worsened outcome; however, further prospective validation studies are needed to support the here presented preliminary results.


| INTRODUC TI ON
Seasonal influenza is an important cause of morbidity and mortality worldwide, with an annual incidence rate estimated between 5% and 10% in adults and 20% and 30% in children, resulting in about 290 000-650 000 deaths per year. 1 Especially infants, older adults and patients with chronic illnesses like cardiovascular disease or immunosuppression are at risk. 2,3 Continuous variations of circulating seasonal influenza virus subtypes, dual or repeated infections during one season and limited effectivity of vaccination are major challenges. [4][5][6] The pteridine neopterin is a marker of cell-mediated immune activation, which is biochemically inert and whose half-life in the human organism is mainly affected by renal excretion. [7][8][9] Activation of monocytes/macrophages after stimulation with interferon-gamma (IFN-γ) leads to increased production of neopterin. 8,9 However, activation of the immune system via IFN-γ also results in increased tryptophan catabolism through the kynurenine pathway. Degradation of tryptophan to kynurenine is catalyzed by indoleamine 2,3-dioxygenase (IDO). 10 Indoleamine 2,3-dioxygenase is inducible by pro-inflammatory cytokines, in particular IFN-γ. [11][12][13] Increased neopterin levels are observed during viral infections, and previous studies have shown that serum neopterin levels combined with C-reactive protein (CRP) can differentiate between viral and bacterial etiologies in acute respiratory tract infections. [14][15][16] Increased levels of neopterin have previously also been associated with adverse outcomes in different diseases including viral infections. 8,[16][17][18] Similarly, tryptophan metabolism is able to slow down cellular immune response, for example, during infections, and thus, the determination of tryptophan metabolites may hold promise to predict outcomes in viral infections. 19 Since influenza antigen bed-side tests have limited sensitivity and polymerase chain reaction (PCR)-based methods are expensive and often not available, cheaper and more reliable biomarkers for diagnosis, and risk stratification of patients with influenza infection are desired. 20 The aim of this pilot study was to investigate neopterin levels and IDO activity in sera of hospitalized patients with influenza infection and to analyze correlations with clinical outcome parameters in a small real-life cohort. We also questioned if discrimination between influenza and community-acquired pneumonia (CAP) is possible by assessing neopterin serum levels.

| Subjects
We retrospectively analyzed eligible subjects by reviewing the electronic medical records of patients, who were treated at the

| Clinical cases
Forty patients were included in this retrospective analysis, and the ratio of influenza A and B was 3:  Figure 2). When splitting the cohort into patients with either influenza virus type A or B, no significant differences between laboratory parameters were seen (Table 2).
Neopterin levels significantly correlated with creatinine (r = .34, P = .03) and hsTroponinT (r = .47, P < .01), while no significant correlations were found with CRP and WBC. The mean length of F I G U R E 1 Algorithm for the inclusion of patients

| Tryptophan metabolism
Fourteen serum samples of patients with confirmed influenza infection (Flu A: Flu B = 13:1) were identified in our biobank and subsequently analyzed and compared to a healthy historical cohort. The exact details of the control cohort are described elsewhere. 22 The mean concentrations of neopterin, tryptophan, kynurenine, and Kyn/Trp, as well as the demographic details of the sub-cohort, are shown in Table 3. Overall, we found significantly higher levels of neopterin (P < .01), kynurenine (P < .01), Kyn/Trp (P < .01), and lower levels of tryptophan (P < .01) when comparing influenza patients to healthy controls (Figures 2 and 3).
Positive correlations were found between neopterin and kynurenine (r = .53, P < .01) as well as between neopterin and Kyn/Trp

| The discriminative potential of neopterin in the setting of influenza and CAP
To analyze the discriminative potential of neopterin, a cohort of 48 patients with CAP was compared to the influenza patients. 23 The two cohorts were well matched in terms of age, gender, and creatinine levels ( Table 1). Community-acquired pneumonia and WBC concentrations were significantly higher in CAP patients compared to influenza patients (P < .01), whereas neopterin concentrations were significantly lower (P < .01, Table 1, Figure 2). Receiver operating characteristic analysis for differentiation between CAP and influenza patients showed the highest AUC for CRP (AUC = 0.849), followed by WBC (AUC = 0.680) and neopterin (AUC = 0.663, Figure 4).

| D ISCUSS I ON
Influenza is an important cause of morbidity and mortality worldwide. Several inflammatory markers, which correlate with disease severity, have intensively been studied in the past.
In our pilot study, we were able to show an elevation of neopterin

| CON CLUS ION
In summary, we were able to show an elevation of neopterin serum levels as well as a simultaneous induction of IDO as represented by an elevated Kyn/Trp in all patients with influenza virus infection.
Neopterin seems to be related to outcome variables, while in our small cohorts, the performance to differentiate between CAP and influenza was poor. Neopterin might be a valuable biomarker to identify patients at an elevated risk of a worsened outcome; however, further prospective validations studies are needed to support the here presented results.

Support by the "Verein zur Förderung von Forschung und
Weiterbildung in Infektiologie und Immunologie an der Medizinischen Universität Innsbruck" is gratefully acknowledged.
The author(s) received no specific funding for this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are within the paper. The data used to support the findings of this study are available from the corresponding author upon request (rosa.bellmann-weiler@i-med.ac.at). Data cannot be shared publicly because of privacy concerns.