Immunogenicity of protein aldehydes

Protein carbonylation introduces diverse non-enzymatic post-translational modifications (PTMs) containing aldehydes and ketones (reactive carbonyls) during oxidative or glycative stress, and is a hallmark of unhealthy ageing and various neurological, cardiovascular, respiratory, musculoskeletal and metabolic diseases. The activation of innate and adaptive immune responses to carbonylated proteins is thought to trigger or exacerbate these pathologies, although how specific PTMs activate the immune system is not well understood. In this thesis, the protein hen egg lysozyme (HEL) was modified with glycolaldehyde (GA) as a model to investigate the immunogenicity of protein aldehydes. Mice immunised with HEL GA in the absence of adjuvant produced higher IgG titres to native HEL protein. Chemical reduction of aldehydes to non-reactive alcohol groups maintained equivalent levels of protein cross-linking and mostly abrogated immunogenic responses. Moreover, mice immunised with GA-modified self-homologue mouse lysozyme (ML) produced anti-ML IgG responses to break self-tolerance. T and B cell adaptive immune responses were investigated to determine their role in GA-mediated immunogenicity. Antigen-specific CD4+ T cell recall responses to native HEL protein were not elevated in mice immunised with HEL GA compared with controls. In contrast, the number of HEL-specific antibody secreting cells (ASCs) was increased in mice after HEL GA immunisation. Innate immune responses were then considered as potential factors mediating enhanced GA-protein immunogenicity. Binding assays showed that HEL GA had increased mouse C3 degradation product opsonisation compared with controls. Mice depleted of complement C3 using cobra venom factor then immunised with HEL GA suggested greater early anti-HEL IgG responses compared with non-depleted mice. C3 knockout mice immunised with HEL GA also appeared to show increased anti-HEL IgG responses, suggesting an immunoregulatory rather than immunoenhancing role for complement in GA-mediated immunogenicity. Pull-down and co-immunoprecipitation experiments to determine whether GA-modified proteins could engage innate immune factors confirmed increased complement activation and immunoglobulin binding. In summary, protein aldehydation induced by GA treatment increases IgG anti-body responses to native protein and can break self-tolerance in the absence of adjuvant. Protein aldehydes are confirmed to be the immunogenic moiety underlying this immunogenicity because their chemical reduction abrogates most of the ensuing antibody response. Protein aldehydes appear to induce greater B cell activation and differentiation to ASCs, although the GA-enhanced B cell response does not seem to be mediated by CD4+ T helper cells. The mechanism underlying GA-enhanced immunogenicity remains to be determined, although protein aldehydes have altered interactions with various soluble factors including complement and immunoglobulin. This thesis adds to existing knowledge about how protein aldehyde PTMs may activate innate and adaptive immune responses to ultimately lead to unhealthy ageing and associated pathologies.