AbstractAlzheimer’s disease (AD) is a neurodegenerative condition affecting approximately 30 million people worldwide. The abnormal build-up of amyloid beta (Aβ) peptide has been implicated in AD and is thought to cause damage to neurons and inflammation contributing to disease progression. In addition to the ‘normal’ Aβ there are also a number of post translationally modified (PTM) variants of the peptide. It is thought that the immune system is able to recognise Aβ, through different immune receptors including toll-like receptors (TLR)2 and 4 and T-cell receptors (TCRs), contributing to both inflammation and clearance of Aβ from the brain. There is a possibility that the PTM variants of Aβ will cause an altered immune response. Unmodified Aβ peptide and three PTM variants were tested, their ability to aggregate was investigated and their morphology was determined using transmission electron microscopy (TEM). ELISA was used to investigate the release of cytokines in response to the four Aβ peptides from human embryonic kidney293 (HEK293) cells expressing TLR2 or TLR4-MD2-CD14, primary human monocytes and peripheral blood mononuclear cells (PBMCs) from patients with AD and age-matched controls. A cohort of 40 patients with AD and 40 age-matched controls was recruited to investigate antigen-specific T-cell responses to the peptides using flow-cytometry to measure proliferation and phosphorylation of protein kinase C (PKC)-ζ and PKC-δ. TEM analysis of the Aβ peptides showed that they were all able to form mature fibrils although the modified Aβ peptides had a fibrillar morphology earlier than the unmodified form. IL-8 was detected in the supernatants of HEK293 cells expressing TLR2 but not TLR4 after stimulation with fibrillar Aβ (fAβ) peptides and there were no differences observed between unmodified and PTM variants. No IL-1β or TNFα was detected from isolated monocytes but IL-1β was released from PBMCs after stimulation with fibrillar forms of all the peptides tested, the use of flow cytometry showed that the IL-1β was released from monocytes. The addition of neutralising antibody for TLR2 was able to reduce the IL-1β signal in some participants but not in others. In contrast, the use of a second batch of the same peptides was unable to induce a response from PBMCs. It was not possible to see Aβ-specific activation of T-cells leading to proliferation, or phosphorylation of PKC-δ or PKC-ζ. These results suggest that modifications to the Aβ peptide are able to alter the aggregative properties of the peptide but they do not change the immune responses observed. Fibrillar Aβ peptide and its PTM variants can be recognised by TLR2, however other mechanisms are likely to be involved in cytokine release. For example, T-cells may be able to influence the release of cytokines from monocytes. Published data looking at Aβ-specific T-cell activation shows varied results, in this study it was observed that unaggregated Aβ peptide was not able to activate antigen-specific T-cells. There is, however, a possibility that an aggregated form of the peptide would have an effect on T-cells. It was also clear in this study that different batches of peptide are able to induce different biological responses.
|Date of Award||Feb 2016|
Immune responses to amyloid beta and post-translationally modified variants
Whiteley, L. (Author). Feb 2016
Student thesis: Doctoral Thesis