Immune system modulation by advanced glycation end products and their reactive intermediates

Advanced Glycation End Products (AGEs) are a heterogenous group of protein modifications generated in a series of complex chemical reactions which begin with a non-enzymatic reaction between a reducing sugar and a free amine group. AGEs can be formed during food processing, for example, during dry roasting of peanuts. AGEs can also be formed endogenously, including during inflammation. AGE- modification can change protein recognition by the immune system. AGE-modified proteins can bind to scavenger receptors including scavenger receptor A (SR-A), a receptor expressed by macrophages and dendritic cells (DCs) which mediates internalisation of ligands. AGEs can also bind to the receptor for advanced glycation end products (RAGE), a receptor which promotes inflammation. Studies have also suggested that AGE-modified proteins can activate complement, the humoral component of the innate immune system. AGEs are reported to enhance the immunogenicity of modified proteins, that is to increase their capacity to induce adaptive immune responses. However, the mechanisms underlying this phenomenon remain unclear.
In this thesis, a variety of different AGE-modified proteins were created, the modifications characterised and the capacity to induce complement activation assessed. These were then used to immunise mice, including mice lacking SR-A, MARCO (another scavenger receptor of the same family as SR-A), or RAGE. The development of immune responses to native protein was then assessed by measuring antibody responses and performing splenocyte restimulation studies. Murine bone marrow derived DCs (BMDCs) were used to assess AGE-induced maturation of DCs and uptake of AGE-modified proteins. For the latter experiments DCs were also derived from mice lacking SR-A and/or MARCO. Lastly, proteins were also isolated from dry roasted (DR) and raw peanuts. These were characterised, assessed for DC uptake and activation and used to immune mice. The results demonstrated that glucose (GLUC)-modified hen egg lysozyme (HEL) induced significant anti-HEL splenocyte responses but no detectable IgG responses. In contrast, modification with glycolaldehyde (GA), an intermediate in AGE formation, induced significant antibody responses without significant splenocyte responses. GA-modified, but not GLUC-modified, HEL induced complement activation in ELISA-based studies. None of the tested AGE samples induced DC activation. However, GLUC-modification significantly enhanced BMDC uptake of proteins. In contrast to previous studies, this uptake was not dependent on the scavenger receptor SR-A. Indeed immunisation studies using knockout (KO) mice indicated that the receptors SR-A, MARCO and RAGE were all dispensable for the formation of GLUC-induced immune responses. However, these studies did reveal that mice lacking MARCO, particularly SR-A/MARCO double KO, gain excess body fat with age. In contrast, RAGE KO mice has lower body fat than WT controls and immune cells isolated from lymph nodes, spleen, liver and adipose tissue of RAGE KO mice were autofluorescent. The proteins isolated from DR peanuts showed low levels of AGE-modification and did not induce DC maturation or have greater DC uptake. Immunisation studies with the peanut proteins did not show that DR peanut proteins have significantly greater immunogenicity than raw peanut proteins.
To summarise, different AGE-modified proteins induce contrasting immune responses which appear to be mediated by different mechanisms. As complement deposition is known to enhance B cell responses to proteins, GA-modification may induce antibody production through complement activation. The enhanced immunogenicity of the GLUC-modified samples may be the result of increased DC uptake, however the enhanced responses do not require MARCO, SR-A or RAGE. This thesis also revealed roles for MARCO, RAGE and SR-A in regulating body adiposity, a finding which may be of significant interest with regards to obesity. However, the studies included here suggest that DR peanut proteins do not have greater immunogenicity than raw peanut proteins in murine immunisation models.

Keywords: Advanced Glycation End Products
Dendritic Cells
Complement
Scavenger Receptor A
Glycolaldehyde
MARCO
RAGE
Advisor: Sattentau, Q