Background

Multiple lines of evidence support peripheral organs in the initiation or progression of Lewy body disease (LBD), a spectrum of neurodegenerative diagnoses that include Parkinson’s Disease (PD) without or with dementia (PDD) and dementia with Lewy bodies (DLB). However, the potential contribution of the peripheral immune response to LBD remains unclear. This study aims to characterize peripheral immune responses unique to patients with LBD at single-cell resolution to highlight potential biomarkers and increase mechanistic understanding of LBD pathogenesis in humans.

Methods

In a case-control study, peripheral mononuclear cell (PBMC) samples from research participants were randomly sampled from multiple sites across the United States. The diagnosis groups comprise healthy controls (HC, n=159164), LBD (n=110132), Alzheimer’s disease dementia (ADD, n=9798), other neurodegenerative disease controls (NDC, n=1921), and immune disease controls (IDC, n=14). PBMCs were activated with three stimulants (LPS, IL-6, and IFNa) or remained at basal state, stained by 13 surface markers and 7 intracellular signal markers, and analyzed by flow cytometry, which generated 1,184 immune features after gating. 

Results

The model classified LBD from HC with an AUROC of 0.8790±0.06 and AUPRC of 0.806±0.06. Without retraining, the same model was able to distinguish LBD from ADD, NDC, and IDC. Model predictions were driven by pPLCγ2, p38, and pSTAT5 signals from specific cell populations under specific activation. The immune responses characteristic for LBD were not associated with other common medical conditions related to the risk of LBD or dementia, such as sleep disorders, hypertension, or diabetes.

Conclusions and Relevance

Quantification of PBMC immune response from multisite research participants yielded a unique pattern for LBD compared to HC, multiple related neurodegenerative diseases, and autoimmune diseases thereby highlighting potential biomarkers and mechanisms of disease.

PBMCs were isolated by density-gradient centrifugation and cryopreserved. Post-thaw, cells were washed in a complete RPMI medium with benzonase. Cell viability as measured by Vi-Cell (Beckman Coulter) for all samples was above 90%. After resting for 2h at 37oC, PBMCs were either left unstimulated or stimulated with a panel of cytokines: IFNα (10,000 units/ml), IL-2 (50 ng/ml), IL-6 (50 ng/ml) and LPS (200 ng/ml) for 15 min, at 37oC. Stimulation was stopped by fixing cells with paraformaldehyde for 10 minutes at room temperature. After washing cells with PBS, samples were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation (from Invitrogen) for 15 min at room temperature. After live dead staining, cells were washed with wash buffer (Phosphate buffered saline, 2% Fetal bovine serum, 0.1% sodium azide), followed by surface staining with anti- CD4(BUV805), CD7 (AF780), CD8 (AF700), CD11b (BUV395), CD14 (BUV737), CD16 (BV750), CD19 (PerCP-Cy5.5), CD27 (BV711), CD56 (BUV563), CD69 (BUV661), HLA-DR (BV480) (antibodies from BD Biosciences), CD3 (BV605) and CD45RA (BV570) (antibodies from BioLegend). Staining was done at room temperature for 30 min. After 2 washes, cells were permeabilized with ice-cold methanol and were stored overnight at -80°C. Post permeabilization, cells were washed again, and intracellular staining was done with anti-pSTAT1 (AF488), pSTAT5 (PE-Cy7), pP38 (PE), pPLCγ2 (APC), pS6 (BV421), CD107b/Lamp2 (BV786), (antibodies from BD Biosciences) and Rab5 (PE-CF594) (from Santa Cruz Biotechnology) at room temperature for 30 min. After two further washes, the acquisition was performed on a BD Symphony A5 flow cytometer with a High Throughput Sampler (HTS) and analyzed using FlowJo software.