The Importance of Macrophages, Lipid Membranes and Seeding in Experimental AA Amyloidosis

Sammanfattning: Amyloidosis is a group of protein misfolding diseases caused by tissue deposition of fibrillary protein aggregates termed amyloid. Amyloid A (AA) amyloidosis is a systemic form of amyloidosis that occurs as a complication of chronic inflammatory diseases, such as rheumatoid arthritis, familial Mediterranean fever and chronic infections, such as tuberculosis. AA amyloid is derived from the precursor protein serum amyloid A and is deposited in several organs preferably kidneys, liver and spleen. AA amyloidosis can be induced in mice by long standing inflammatory stimulation and concurrent administration of tissue extracts of AA amyloid, referred to as amyloid enhancing factor (AEF), reduces the time for amyloid deposition in the marginal zone of the spleen from 5 weeks to 2 days. The general aim of this thesis was to investigate the mechanisms involved in the development of AA amyloid in the mouse model of AA amyloidosis.Amyloid was induced in inflamed mice by injection of AEF and amyloid toxicity to splenic macrophages was investigated. We found that the marginal zone macrophages were very sensitive to amyloid formation and increasing amyloid load caused progressive depletion of these cells, whereas red pulp macrophages and metallophilic marginal zone macrophages appeared unaffected. To clarify the role of splenic macrophages in amyloidogenesis, macrophages were depleted by clodronate containing liposomes. We displayed that in the absence of splenic macrophages, especially marginal zone macrophages, amyloid formation was delayed implying a crucial role of macrophages in amyloid formation.The effect of lipid membranes on amyloid formation was studied and we showed that liposomes exhibited an amyloidogenic effect in inflamed mice although not as powerful as AEF. Following the fate of the liposomes, we showed that liposomes were rapidly cleared by uptake in the spleen and liver and colocalized with lysosomes. A tentative mechanism might be that accumulation of liposomes in lysosomes interfere with the SAA degradation process facilitating amyloid formation.Finally the conformational properties of two AEF (AEF1 and AEF2) preparations were studied using conformation sensitive luminescent-conjugated oligothiophenes (LCOs). We found that AEF1 and AEF2 displayed significantly different ultrastructure as well as conformation and consequently induced different cytotoxicity in vitro. Inducing amyloid formation in inflamed mice by AEF1 and AEF2 revealed that the polymorph of the amyloid aggregates was replicated in vivo.In summary, the results obtained in this thesis indicate an important role for macrophages for the formation of amyloid. The existence of amyloid strains has long been an in vitro finding, but the finding that AEF ultrastructure drives the morphology of newly formed amyloid in vivo opens up for new studies that can help us to understand the formation of homologous and heterologous fibrils. Thus, the fundamental mechanisms of various amyloid diseases are similar and the results presented in the thesis can increase the understanding of other amyloid diseases.