T-cell dysregulation is associated with disease severity in Parkinson's Disease

Abstract

The dysregulation of peripheral immunity in Parkinson's Disease (PD) includes changes in both the relative numbers and gene expression of T cells. The presence of peripheral T-cell abnormalities in PD is well-documented, but less is known about their association to clinical parameters, such as age, age of onset, progression rate or severity of the disease. We took a detailed look at T-cell numbers, gene expression and activation in cross-sectional cohorts of PD patients and age-matched healthy controls by means of flow cytometry and NanoString gene expression assay. We show that the well-pronounced decrease in relative T-cell numbers in PD blood is mostly driven by a decrease of CD8⁺ cytotoxic T cells and is primarily associated with the severity of the disease. In addition, we demonstrate that the expression of inflammatory genes in T cells from PD patients is also associated with disease severity. PD T cells presented with increased activation upon stimulation with phytohemagglutinin that also correlated with disease severity. In summary, our data suggest that the consequences of disease severity account for the changes in PD T cells, rather than age, age of onset, duration or the disease progression rate.

Keywords: Disease severity; Gene expression; PHA; Parkinson’s Disease; T cells.

Fig. 1

Decrease of total CD3⁺ T cells in PD is associated with disease progression. A, B Flow-cytometric analysis of peripheral blood leucocytes from HC (n = 20, f/m = 13/7) and PD patients (n = 36, 12/24 f/m) reveals a significant decrease of total CD3⁺ T cells (as % of all leucocytes) in PD. C, D T cells are decreased in PD in both age groups (≤ 64 years and > 64 years) and in both sexes (two-way ANOVA: case **p < 0.01, sex p > 0.36, age p > 0.35, no statistically significant interactions). E T-cell decrease correlates significantly with disease progression (Spearman’s ρ). F–I CD3⁺ T cells are significantly decreased in PD patients with advanced disease progression (H&Y ≥ 3) (I), but not earlier age at onset (F), longer disease duration (G), or higher progression rate (H, H&Y/year). Boxplots: median ± interquartile range; *p < 0.05, **p < 0.01, Mann–Whitney U test, two-way ANOVA

Fig. 2

CD4⁺ and CD8⁺ T cells are decreased in PD. A CD3⁺CD4⁺ T cells are significantly decreased in PD patients (as % of all leucocytes, two-way ANOVA: case *p < 0.05, sex p > 0.36, age p > 0.41, no statistically significant interactions). B Correlation of CD3⁺CD4⁺ cell numbers with PD clinical traits (Spearman’s ρ). C, D CD3⁺CD4⁺ cell numbers in PD patients stratified by disease duration (C) and disease progression (D). E CD3⁺CD8⁺ T cells are significantly decreased in PD patients (as % of all leucocytes, two-way ANOVA: case **p < 0.01, sex p > 0.41 age p > 0.62, no statistically significant interactions). F Correlation of CD3⁺CD8⁺ cell numbers with PD clinical traits (Spearman’s ρ). G, H CD3⁺CD8⁺ cell numbers in PD patients stratified by disease duration (G) and disease progression (H). Boxplots: median ± interquartile range; *p < 0.05, ****p < 0.0001, Mann–Whitney U test, two-way ANOVA

Fig. 3

Expression of inflammatory genes in T cells from PD patients is associated with disease progression and disease duration. A Expression of 249 inflammatory genes was quantified with a NanoString expression assay in isolated CD3⁺ T cells from HC (n = 14, f/m = 10/4) and PD patients (n = 16, f/m = 4/12). Mean expression of all 249 quantified transcripts was slightly increased in PD patients (p = 0.24). B PD patients could not be identified based on the expression signature of all 249 transcripts (complete linkage with Euclidean distance). C Volcano plot and heatmap (D) of the differential expression analysis between HC and PD T cells. Nine genes were significantly differentially expressed in PD T cells (fdr q < 0.05, fold change < − 1.4/> 1.4). None of the nine genes’ expression was significantly affected by the age or sex of participants (two-way ANOVA). E Significant up-regulation of selected transcripts could be verified in an independent validation cohort by RT-qPCR (HC/PD n = 12/12). F WGCNA analysis reveals six blocks (modules “A”–“F”) of genes, which are co-expressed in a similar fashion amongst all samples. The weighted co-expression scores of these modules do not correlate significantly with case (HC or PD), sex, age or age at disease onset. Three gene modules (“B”, “E” and “F”) significantly correlated with disease duration and disease progression in PD patients. G GO enrichment analyses of modules “B”, “E” and “F” against the full list of all 249 quantified inflammatory genes reveals association of disease progression with genes important in acute inflammatory response and gliogenesis, and of disease duration with I-κB/NF-κB signaling. Boxplot: median ± interquartile range; lines (E): median; heatmap rows (genes) scaled to z-scores; *p < 0.05, Mann–Whitney U test; WGCNA correlation assessed with Pearson’s correlation coefficient; GO fdr q < 0.05

Fig. 4

Increased activation of PD T cells by PHA is associated with disease progression. A, B CD3⁺ T cells were enriched from peripheral blood, stimulated with PHA and T-cell activation assayed by quantitation of CD69 expression on the cell surface. Activation was slightly increased in PD patients’ T cells [HC (n = 14, f/m = 6/8) and PD patients (n = 23, 6/17 f/m), p < 0.09, two-way ANOVA: case p < 0.10, sex p > 0.99, age p < 0.16]. C Correlation of T-cell activation with PD clinical traits (Spearman’s ρ). D T-cell activation is significantly increased in PD patients with advanced disease progression (H&Y ≥ 3. two-way ANOVA: H&Y *p < 0.05, sex p > 0.25, age p > 0.51). Boxplots: median ± interquartile range; *p < 0.05, Mann–Whitney U test, two-way ANOVA