Short amyloid-beta immunogens show strong immunogenicity and avoid stimulating pro-inflammatory pathways in bone marrow-derived dendritic cells from C57BL/6J mice in vitro
Highlights
• Short Aβ peptides increase migration/endocytosis of immature DCs. • Short Aβ peptides increase MHC II molecule expression and alloreactive T cell activation in TNF-α-matured DCs. • Short Aβ peptides exhibit decreased production of Th1/Th2 cytokines and pro-inflammatory cytokines.
Introduction
The pathological hallmarks of Alzheimer's disease (AD) are intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein and extracellular senile plaque deposits, comprised mainly of amyloid beta (Aβ) peptide. Aβ, the main constituent of amyloid plaques, is believed to be the main causative factor of AD pathogenesis [11], [21]. Aβ is formed after sequential cleavage of the amyloid precursor protein by β- and γ-secretases. The most common isoforms are Aβ40 and Aβ42.
Aβ immunotherapy has attracted significant attention since the first dramatic success in a mouse model of AD [29]. Initial human trials of active Aβ1–42 vaccination (AN1792), however, were halted early because of meningoencephalitis in 6% of subjects [25]. The failure might be ascribed to a promiscuous T cell epitope, which is located within Aβ15-42 and known to activate Aβ specific T cells [8], [22], [23], [39]. Short immunogens, which include B cell epitopes but lack T cell epitopes, such as Aβ1–15 and its derivatives, have shown promise as immunogens. Recent studies suggest that Aβ1–42 modulates the immune system by inducing pro-inflammatory dendritic cells (DCs) with reduced antigen-presenting function [4], [5]. It was therefore of interest to investigate how Aβ1–15 or its derivatives would impact DCs function.
We tested two immunogens, which were a tandem repeat of two-lysine-linked Aβ1–15 sequences (P1) with the addition of an Arg-Gly-Asp (RGD) motif (P2). The aim of this study was to analyze safety and immunogenicity of the peptides by examining DC function.
Section snippets
Mice and reagents
The Nanjing Medical University Experimental Animal Care and Use Committee approved the protocol. The Laboratory Animal Center of Nanjing Medical University provided wild-type C57BL/6 mice (4–6 weeks old). The following reagents were used: RPMI-1640, Fetal Bovine Serum, Penicillin, streptomycin (Hyclone, Logan, UT, USA), recombinant mouse GM-CSF, recombinant mouse TNF-α, IL-4 (PeproTech, Rocky Hill, NJ, USA), FITC-Dextran (Sigma–Aldrich, St. Louis, MO, USA), FITC anti-mouse CD11c (Integrin αX,
Both P1 and P2 do not decrease the viability of DCs
First, we investigated the purity of DCs by flow cytometric analysis of CD11c expression. The purity of BMDCs obtained from this method could reach 95% (Fig. 1A). The occurrence of survival and/or apoptosis in DCs (con-DCs, P1-DCs, P2-DCs) was assessed by the MTT assay or by Annexin V/PI method. As shown in Fig. 1B, neither P1 nor P2 lowered cell viability. When cells were analyzed by flow cytometry and the Annexin V/PI method, peptides did not appear to induce apoptosis (Fig. 1C). Together,
Discussion
The present study demonstrates that two new peptides, which lack T cell epitopes but include B cell epitopes, are able to modulate the function of BMDCs from C57Bl/6J mice. A key question is how short Aβ immunogens modulate the function of DCs. Both peptides promote migration and endocytosis of immature DCs, and increased MHC class II expression/alloreactive T cell activation in TNF-α-matured DCs. At the same time, they decreased the production of Th1/Th2 regulatory and pro-inflammatory
Conclusions
In conclusion, we investigated the interactions between short Aβ immunogens and DCs in vitro. Both P1 and P2 enhance migration/endocytosis of immature DCs and increase MHC class II expression/alloreactive T cell activation in TNF-α-matured DCs. In addition, shorter Aβ immunogens could reduce Th1 pro-inflammatory cytokines and increase anti-inflammatory cytokines.
Author's contributions
JX, YDZ, JPS and HX participated in concept and design of the study. JQS, BRW, JC, YWZ, YX and JW participated in acquisition of raw data, analysis and interpretation of data, drafting manuscript. JX and YDZ participated in critical revision of the manuscript.
Acknowledgments
This work was supported by a National Natural Science Foundation of China grant (30700248). We thank the Laboratory of Neurotoxicology of Nanjing Medical University for help with cell culture. The authors declare that they have no competing financial interests.
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