Selected serum cytokines and nitric oxide as potential multi-marker biosignature panels for Parkinson disease of varying durations: a case-control study

Abstract

Background: Dopaminergic neuronal loss begins years before motor symptoms appear in Parkinson disease (PD). Thus, reliable biomarkers for early diagnosis and prognosis of PD are an essential pre-requisite to develop disease modifying therapies. Inflammation-derived oxidative stress is postulated to contribute to nigrostriatal degeneration. We evaluated the role of selected serum immune mediators (IFNγ, TNFα, IL-10, and NOx) in PD progression and estimated their usefulness in preclinical diagnosis.

Methods: This case-control study recruited 72 PD patients with varying disease durations (< 1-year, n = 12 patients; 1-3 years, n = 30; > 3 years, n = 30) and 56 age- and gender-matched controls (26 with other neurological disorders as disease controls, and 30 healthy controls). Serum cytokine levels and NOx quantified using Sandwich Enzyme Linked Immunosorbent Assay kits, and the Griess test, respectively, were evaluated for diagnostic accuracy of optimal marker combinations by the CombiROC method. PD patients were clinically evaluated for motor and non-motor symptoms, and staged based on Hoehn and Yahr (H-Y) scale.

Results: A significant increase in serum IFNγ and IL-10 was observed in PD compared to healthy controls (p < 0.001). The Th1: Th2 (IFNγ: IL-10) cytokine ratio was higher in PD of 3-12 years compared with PD < 1 year (p < 0.001). Highest levels of NOx manifested during early PD (1-3 years) through a subsequent decline with disease duration. TNFα level was highest at PD onset. A low serum NOx level was associated with cognitive impairment (p = 0.002). The potential of using multi-biomarker panel, IFNγ, IL-10 and TNFα, for detection of PD onset was evident (sensitivity [SE] = 83.3%, specificity [SP] =80.4%, area under curve [AUC] = 0.868), while for early and late PD the multi-biomarker signature of IFNγ, IL-10 and NOx appeared to be more promising (SE = 93.3%, SP = 87.5%, AUC = 0.924).

Conclusion: A Th1 cytokine-biased immune response predominates with PD progression. Both IFNγ and IL-10 are involved in disease severity. However, TNFα-mediated neurotoxicity appears to occur in early PD.

Keywords: Cytokines; Nitric oxide; Parkinson disease; Serum biomarkers; Sri Lanka.

Fig. 1

Concentrations of serum immune mediators in Parkinson patients with varying disease durations compared with controls (a) Mean serum IFNγ concentration in pg/ml (< 1-year PD = 41.76[±3.92], 1–3 years PD = 35.82 [±6.17], > 3 years PD = 51.65 [±8.43]) (b) Mean serum IL-10 concentration in pg/ml (< 1-year PD = 27.97[±5.76], 1–3 years PD = 21.69[±4.29], > 3 years PD = 23.86[±4.21]) (c) Mean serum TNFα concentration in pg/ml (< 1-year PD = 12.43[±6.74], 1–3 years PD = 2.5[±4.04], > 3 years PD = 0.98[±2.53]) (d) Mean serum NOx concentration in mmol/dm³ (< 1-year PD = 10.64[±3.59], 1–3 years PD = 18.47[±2.16], > 3 years PD = 15.26[±5.27]). The results are expressed as mean ± SD. ****P < 0.0001 and ***P < 0.001 were considered significant compared to healthy controls (Mann-Whitney U Test)

Fig. 2

Th1:Th2 serum cytokine ratio (IFNγ:IL-10) in PD patients of varying disease durations, and in controls. Each Th1:Th2 ratio was calculated using individual serum concentrations of IFNγ and IL-10 to obtain the mean ratio of each PD patient for statistical analyses. (Mann-Whitney U Test *P < 0.0001)

Fig. 3

ROC curves for comparing multiple markers and their combinations in PD groups of varying durations.(a) less than 1 year PD, (b) 1–3 years of PD and (c) 3–12 years of PD. ROC curve depicts true positive rate (sensitivity [SE]) on y-axis as function of false positive rate (1 – specificity [SP]) on x-axis. Higher y values correspond to higher SE, whereas lower x values correspond to higher SP

Fig. 4

Density distribution of permutated AUC values compared to the normal distribution. The significance of real AUC value for (a) less than 1 year of PD, (b) 1–3 years of PD and (c) 3–12 years of PD. The‘real AUC values’ lie outside the permutated AUC distribution that symbolise high validity of the marker panels generated via CombiROC