The Role of ASC in the Pathogenesis of Systemic Amyloid A Amyloidosis

The innate immune system plays a fundamental role in health and disease. The apoptosisassociated speck-like protein containing a caspase recruitment domain (ASC) protein is a component of ASC inflammasomes, inflammation-mediating complexes that include the Nucleotide-Binding Oligomerization Domain, Leucine Rich Repeat And Pyrin Domain Containing 3 (NLRP3) inflammasome. ASC and NLRP3 inflammasome components aggregate to form ASC specks, which can bind β-sheet rich proteins and cross-seed Aβ aggregation in Alzheimer’s disease (AD). Therefore, it is conceivable that ASC recognizes other β-sheet rich and amyloidogenic proteins involved in additional protein misfolding diseases (PMDs), such as systemic amyloid A (AA) amyloidosis. AA amyloidosis is a severe complication of chronic inflammatory conditions and is caused by the aggregation of the major acute-phase protein Serum Amyloid A (SAA). In systemic AA amyloidosis, abnormally folded AA proteins aggregate in the extracellular compartment and cause organ dysfunction and failure due to disruption of tissue integrity. Since AA and Aβ aggregates share similar biochemical properties, such as β-sheet structures, we hypothesized that ASC inflammasomes may be involved in SAA/AA recruitment, aggregation, processing and removal. To date, the involvement of inflammasomes in SAA recruitment and AA fibril formation has not yet been described. Furthermore, no therapeutic approaches that directly interfere with SAA recruitment or AA amyloid formation are available, meaning that treatment of systemic AA amyloidosis primarily aims to reduce the underlying inflammatory state. In the work presented in this thesis I investigated the role of ASC in AA amyloidosis in vivo, employing a murine AA amyloidosis model in Asc-ablated (Asc-/-) mice with the prospect that ASC may emerge as potential therapeutic target to fight systemic AA amyloidosis. To do so, I targeted the following objectives. First, I challenged Asc-/- mice with silver nitrate, the proinflammatory reagent used in the murine model of AA amyloidosis. As of now, it has not been described whether silver nitrate is sensed as danger signal by ASC-dependent inflammasomes (Eisenbarth, Colegio et al. 2008, Lundmark, Vahdat Shariatpanahi et al. 2013). If this were the case, an in vivo study harnessing genetically Asc-ablated mice, would be impossible since an acute-phase response, reflected by SAA serum protein elevation, is prerequisite for the development of AA amyloidosis. I was able to show that Asc wildtype (wt) and Asc-/- animals that had been injected with silver nitrate only did not exhibit a significant difference in the mean SAA serum peak concentrations at 24 hours after subcutaneous injection of silver nitrate. The fact that genetic ablation of Asc does not significantly impact silver nitrateinduced SAA levels excludes a major contribution of ASC inflammasomes in silver nitrate sensing and makes the murine AA amyloidosis model suitable for in vivo studies harnessing Asc-/- mice. In a second step I performed an in vivo study using the murine model of AA amyloidosis and genetically ablated Asc-/- mice. As this PMD model necessitates an accurate acute-phase (immune) response, I did not want broad sex and age differences between experimental animals that could potentially entail varying immune responses. Therefore, I decided to perform the in vivo study in F2 generation littermates with minimal interindividual sex and age difference. This served to reduce potential biases caused by e.g. sex differences, microbiome variancerelated changes in immune (inflammatory) response (Maynard and Weinkove 2018, Thevaranjan, Puchta et al. 2018) and age-related differences in immune response, also referred to as “inflammaging” (Franceschi, Bonafè et al. 2000, Frasca and Blomberg 2016, Criscuolo, Sorci et al. 2018). A total of 79 animals met the criteria for study inclusion. Since the acute-phase reactant SAA is the precursor of fibrillogenic AA proteins (Prelli, Pras et al. 1987, Tape, Tan et al. 1988, Kluve-Beckerman, Manaloor et al. 2002), I assessed the temporal development of SAA serum concentrations in a next step, in order to draw conclusions about SAA recruitment (from serum to the tissue) in AA amyloidosis pathogenesis – in absence and presence of ASC. To address this question, I drew blood before as well as up to four days after injections of silver nitrate and/or amyloid enhancing factor (a preparation of AA fibrils). I showed that the longitudinal development of SAA serum levels differed significantly between Asc wt and Asc-/- mice with AA amyloidosis. Asc wt mice exhibited a significant decrease in SAA serum concentrations 24 hours after AA amyloidosis induction, a phenomenon which was abrogated in Asc-/- mice. Furthermore, there was no difference in silver nitrate-only treated mice, suggesting a crucial role of ASC in the recruitment of SAA in the presence of AA fibrils. In a fourth step I determined the splenic amyloid deposition. To do so, I assessed Congo red and luminescent conjugated polythiophene stained spleen sections of experimental mice. Intriguingly, ASC deficient mice with AA amyloidosis showed decreased amyloid deposition and red pulp invasion, suggesting that the ASC protein controls the severity of systemic AA amyloidosis in mice. Moreover, I showed that ASC is incorporated in amyloid deposits of Asc wt mice. As there is still a lack of consensus about how AA amyloidosis affects the cellular architecture in disease state, I wanted to check for potential and disease-specific cellular architecture alterations in the spleen and peripheral blood. Interestingly, there was increased infiltration of innate immune cells in the spleen. Moreover, inflammation decreased the platelet count in Asc wt mice with AA amyloidosis, a phenomenon that was attenuated in Asc-/- mice with AA amyloidosis, revealing a potential role of platelets in inflammatory responses involving ASC inflammasomes and a potential impact in pathogenesis of AA amyloidosis. In a next step I wanted to assess the phagocytic activity of macrophages with different Asc genotypes, as macrophages play a pivotal role in AA amyloidosis. Macrophages co-localize with splenic AA amyloid and clear AA amyloid by immunoglobulin mediated phagocytosis (Nyström and Westermark 2012, Sponarova, Nuvolone et al. 2013). Moreover, AA accumulation is decreased and inhibited upon phagocyte depletion (Lundmark, Vahdat Shariatpanahi et al. 2013, Kennel, Macy et al. 2014). The idea that different expression levels of the ASC protein may result in an altered phagocytosis of macrophages is supported by a recent report that has shown an increased phagocytosis of Asc heterozygous astrocytes in Alzheimer’s disease mice (Couturier, Stancu et al. 2016). Therefore, I applied an in vitro phagocytosis platform harnessing bone marrow-derived macrophages generated from Asc wt, Asc heterozygous and Asc-/- mice. I showed that activated Asc-/- macrophages exert increased phagocytosis, which may point towards a role of ASC in macrophage-mediated AA amyloid processing in AA amyloidosis. Since genetic data presented in this work suggest that the ASC protein controls the severity of systemic AA amyloidosis in mice, I endeavoured, in a final step, to generate a monoclonal anti-ASC antibody derived from an Asc-/- mouse that had been immunized with the ASC protein as an antibody treatment study would complement the genetic data in an orthogonal manner. A suitable and high affinity anti-ASC antibody may therefore be applied in a possible follow-up in vivo study that investigates if AA amyloid formation and deposition is prevented or attenuated by anti-ASC immunotherapy in (Asc) wt mice with AA amyloidosis. Unfortunately, the ASC-specific B cell isolation was ineffective. Nevertheless, another trial may be performed in collaboration with a biotech company. In summary, findings presented in this work may facilitate the opportunity to find specific and promising therapeutic targets to fight AA amyloidosis as well as other entities of systemic amyloidoses that potentially share similar pathophysiological mechanisms