Distinct responses of human peripheral blood cells to different misfolded protein oligomers

Abstract

Increasing evidence indicates that peripheral immune cells play a prominent role in neurodegeneration connected to protein misfolding, which are associated with formation of aberrant aggregates, including soluble protein misfolded oligomers. The precise links, however, between the physicochemical features of diverse oligomers and their effects on the immune system, particularly on adaptive immunity, remain currently unexplored, due partly to the transient and heterogeneous nature of the oligomers themselves. To overcome these limitations, we took advantage of two stable and well-characterized types of model oligomers (A and B), formed by HypF-N bacterial protein, type B oligomers displaying lower solvent-exposed hydrophobicity. Exposure to oligomers of human peripheral blood mononuclear cells (PBMCs) revealed differential effects, with type B, but not type A, oligomers leading to a reduction in CD4⁺ cells. Type A oligomers promoted enhanced differentiation towards CD4⁺ CD25^High FoxP3⁺ Tregs and displayed a higher suppressive effect on lymphocyte proliferation than Tregs treated with oligomers B or untreated cells. Moreover, our results reveal Th1 and Th17 lymphocyte differentiation mediated by type A oligomers and a differential balance of TGF-β, IL-6, IL-23, IFN-γ and IL-10 mediators. These results indicate that type B oligomers recapitulate some of the biological responses associated with Parkinson's disease in peripheral immunocompetent cells, while type A oligomers resemble responses associated with Alzheimer's disease. We anticipate that further studies characterizing the differential effects of protein misfolded oligomers on the peripheral immune system may lead to the development of blood-based diagnostics, which could report on the type and properties of oligomers present in patients.

Keywords: Th1/Th17; Tregs; immunoregulation; neurodegeneration; peripheral immunity; protein misfolding diseases.

FIGURE 1

Comparison of the effects of type A and type B oligomers on human PBMC viability. (a) Evaluation of A and B oligomer cytotoxicity by MTT assay after 24 h of incubation with human PBMCs. PBMC MTT values without exposure to oligomers (control) represented 100% of viability, which is the reference value to calculate the effect as percentage in treated PBMCs. All values are mean ±SEM of eight independent experiments derived from eight healthy donors, each one performed by triplicate. Representative FACS plots are shown in panel b from experiments shown in panel c. Statistically significant differences are indicated as * (p < 0·05) for differences to control cells. (b,c) FACS analysis of the content in CD4⁺ and CD8⁺ T cells after treatment with type A and type B oligomers. (d) FACS analysis of the CD4⁺ T cells after treatment with native HypF‐N protein. Increasing oligomer concentrations in monomers equivalent were added to 1x10⁶ human cultured PBMCs (b‐d). After 24 h of treatment, cells were collected and analysed by FACS as described in Material and methods section. Statistically significant differences are indicated as # (p < 0·05) for differences between type A and type B oligomers at the same concentration or as * (p < 0·005) for differences to control cells

FIGURE 2

Comparison of the effects of type A and type B oligomer exposure on the lymphocyte differentiation to Treg cells and their function in human PBMCs. (a, b) FACS analysis of Treg population (CD4⁺CD25⁺FoxP3⁺) content after 24‐h treatment with increasing concentrations (0·05 μM, 0·5 μM and 5 μM) of type A or type B oligomers (in monomers equivalent). (c) Treg‐mediated suppression of PBMC proliferation after type A or type B oligomer treatment. Sorted Treg cells treated with type A (T_reg Type A Oligomer) or type B oligomers (T_reg Type B Oligomer) or without oligomer exposure (T_reg Control) were cultured at different ratios with concanavalin A‐activated PBMCs (ConA‐activated) or resting PBMCs (Control) as positive or negative controls of cell proliferation, respectively. Representative FACS plots are shown in panel a from eight experiments quantified in panel b. Cells were previously treated in the presence or absence of type A or type B oligomers for 24 h, and regulatory T cells were sorted by FACS. Treg cells were mixed at different ratios with PBMCs in the presence of 2·5 ng/ml ConA and type A or type B oligomers (0·5 μM) for 96 h. Cell proliferation was determined by [³H]‐thymidine incorporation. Percentages represent the average of independent experiments normalized with their control counterparts. All values are mean of eight independent experiments derived from eight healthy donors, each one performed by triplicate. Error bars represent the SEM, and statistically significant differences are marked as ## (p < 0·01) for differences between type A and type B oligomers at the same concentration or as ** (p < 0·01) and *** (p < 0·001) for differences relative to control

FIGURE 3

Interleukin secretion profile of human PBMCs after type A or type B oligomer treatment. (a) Fold changes of proinflammatory cytokines IFN‐γ, IL‐6 and IL‐23. Cytokine basal levels in controls were 4·6 ± 1·6 pg/mL (INF‐γ), 2108 ± 213 pg/mL (IL‐6) and 75 ± 12·7 pg/mL (IL‐23). (b) Fold changes of regulatory cytokines IL‐10 and TGF‐β. Cytokine levels in controls were 3·2 ± 1·8 pg/mL (IL‐10) and 11 ± 4·2 pg/mL (TGF‐β). PBMC cultures without oligomers were used as controls for basal secretion levels. Bars represent the average fold change of eight independent experiments derived from eight healthy donors, each one performed in triplicate. Statistically significant differences are indicated as ## (p < 0·01) for differences between type A and type B oligomers at the same concentration or as * (p < 0·05), ** (p < 0·01) or *** (p < 0·001) for differences related to controls

FIGURE 4

Effects of type A or type B oligomers on Th1 and Th17 lymphocyte differentiation. PBMCs treated for 24 h with type A or type B oligomers or without exposure to oligomers (control) were analysed by intracellular FACS to determine polarization towards Th1 and Th17 subsets. Representative FACS plots are shown in panel a from eight experiments quantified in panel b. Histograms represent the average fold change of eight independent experiments derived from eight healthy donors, each one performed in triplicate. Statistically significant differences are indicated as * (p < 0·05), ** (p < 0·01) or *** (p < 0·001) for differences related to control cells

FIGURE 5

Summary of the functional effects on peripheral immune responses from human PBMC exposure to type A or type B oligomers. The scheme illustrates the differential effects on proinflammatory and regulatory cytokines and Th1/Th17 differentiation and the modelling of AD and PD general features