Abstract
The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) protein is a component of inflammasomes, inflammation-mediating complexes that include Nucleotide-Binding Oligomerization Domain, Leucine Rich Repeat And Pyrin Domain Containing 3 (NLRP3). ASC and NLRP3 components aggregate to form ASC specks, which can cross-seed Aβ aggregation in Alzheimer’s disease (AD). Here we asked whether ASC is involved in additional protein misfolding diseases (PMDs). AA amyloidosis is a severe complication of chronic inflammatory conditions caused by the aggregation of Serum Amyloid A (SAA) protein. 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. We therefore investigated the role of ASC in AA amyloidosis in vivo, employing a murine AA amyloidosis model in Asc-ablated mice. We show that ASC exerts a crucial role in SAA recruitment in the presence of AA fibrils, that splenic AA amyloid load was decreased in Asc-/- mice, that the presence of ASC accelerates SAA fibril formation and that ASC is important for SAA-activated phagocytosis of macrophages. We conclude that ASC modulates both SAA serum levels and the severity of AA amyloidosis in mice. ASC may represent a therapeutic target in AA as well as other amyloidosis entities.
Introduction
In inflammatory conditions, proinflammatory cytokines stimulate hepatocytes to release the acute-phase reactant SAA into the blood stream. During the acute-phase response, the serum concentration of SAA increases as much as 1000-fold from its baseline concentration (1, 2). AA amyloidosis may ultimately develop when persistently high SAA serum concentrations hamper proper SAA processing and clearance, leading to extracellular deposition of aggregated AA fibrils and SAA recruitment to the site of amyloid. AA fibrils are predominantly composed of degradation products of SAA1, an isoform of SAA, which dissociates from its carrier protein high density lipoprotein (HDL) prior to its conversion to amyloid fibrils (3–9).
Amyloid aggregates, composed of AA fibrils and other components, are deposited in the extracellular compartment of the spleen, kidney, liver and heart, which can lead to life-threatening complications due to disruption of tissue integrity (10). Various types of chronic inflammatory conditions, such as chronic bacterial and viral infections (e.g. tuberculosis, intravenous drug abuse, hepatitis B/C), chronic inflammations (e.g. inflammatory bowel diseases, rheumatic arthritis, vasculitis), hereditary mutations (e.g. familial Mediterranean fever or FMF) and neoplastic diseases (e.g. lymphomas) are associated with systemic AA amyloidosis (11, 12). Clinically, systemic AA amyloidosis is often asymptomatic, up to a tipping point in disease progression that heralds rapid health deterioration. At manifestation, AA amyloidosis patients often exhibit proteinuria and progressive kidney dysfunction, followed by liver and heart failure. Current therapies have a limited impact on disease progression and mortality and the exact molecular mechanisms behind the pathogenesis of AA amyloid formation and SAA recruitment remain unclear to date (11, 13). To our knowledge, no therapeutic approaches that directly interfere with SAA recruitment or AA amyloid formation are of avail, meaning that treatment of AA amyloidosis primarily aims to reduce the underlying inflammatory state.
However, recent insights into the field of neurodegenerative PMDs such as Alzheimer’s disease might also be relevant to systemic AA amyloidosis. AD is characterized by the extracellular deposition of aggregates composed of misfolded amyloid β (Aβ) proteins in the central nervous system. Misfolded Aβ proteins contain a larger β-sheet content than soluble Aβ proteins. Similarly, amyloidogenic SAA proteins exhibit a high propensity to form AA fibrils in their misfolded state, where they exhibit increased β-sheet content.
The innate immune system and its components, including inflammasomes, represent the first line of defence to transduce immediate danger signals into a cytokine response, cell death and the activation of the adaptive immune system (14). Furthermore, the innate immune system is involved in the pathogenesis of PMDs (15–17). Upon activation of cytosolic pattern recognition receptors (PRR) of innate immune cells, such as the NACHT-, LRR- and pyrin (PYD)-domain-containing protein 3 (Nalp3) of microglia or macrophages, the adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is recruited, which in turn induces assembly of helical ASC fibrils (18–22). Subsequently, effector caspase-1 is recruited and activated to form mature inflammasomes, enabling them to cleave the proinflammatory cytokine pro-IL1β to IL-1β. ASC fibrils may ultimately transform into large paranuclear ASC specks, helical fibrillary assembly of ASC proteins that can be released from microglia and other tissue-resident macrophages by gasdermin D-mediated pyroptotic cell death (23).
Inflammation and NLRP3/ASC inflammasomes play an eminent role in the pathogenesis of AD (24–26). Intrahippocampal injection of microglia-derived ASC specks leads to Aβ cross-seeding in APP/PS1 Asc wildtype (wt) mice, while cross-seeding and spreading of Aβ pathology was reduced in APP/PS1 Asc-/- mice. Furthermore, stereotactic intrahippocampal co-injection of ASC specks and anti-ASC antibody prevented the increase in Aβ pathology in vitro and in vivo (25, 27).
Intriguingly, several reports point towards a role of ASC in the pathogenesis of AA amyloidosis. For instance, ASC has been shown to co-localize with AA amyloid in kidney biopsies from patients with AA amyloidosis secondary to the hereditary inflammatory condition FMF, where a gain-of-function mutation in the Pyrin gene activates ASC inflammasomes and leads to chronic upregulation and expression of SAA (28). Moreover, SAA activates the NLRP3 inflammasome of human myeloid cells via an interaction with the P2X7 receptor, which induces ASC release in vitro through a cathepsin B-sensitive pathway (29). In line with this finding, SAA (as well as lipopolysaccharide) is capable of activating murine macrophages (30). A murine model of AA amyloidosis has been developed and established. In this model, subcutaneous administration of silver nitrate (AgNO3), coupled with an intravenous application of preformed SAA fibrils (known as Amyloid Enhancing Factor or AEF), rapidly induces the AA amyloidosis phenotype, with respect to splenic AA amyloid deposition (31–33).
AgNO3 serves as a proinflammatory stimulus that leads to elevation in SAA serum levels, while preformed AA fibrils serve as a template for SAA aggregation (34).
The involvement of the innate immune system in AA amyloidosis has been described on a cellular level. For instance, depletion of splenic macrophages delays or inhibits AA amyloid accumulation in mice (31, 35). Furthermore, macrophages were shown to clear AA amyloid via Fc-receptor-mediated phagocytosis (36). However, a role of inflammasomes in SAA recruitment and AA fibril formation has not been described to date.
We, therefore, sought to investigate the role of ASC in the pathogenesis of AA amyloidosis in vivo and assessed the temporal development of AA amyloidosis with a focus on SAA serum concentrations and splenic AA amyloid. Here, we report that Asc wt mice exhibited a pronounced decrease in SAA serum concentration together with a concomitant increase in splenic amyloid load upon AA induction. In contrast, these phenomena were attenuated in the absence of ASC. Furthermore, ASC is incorporated in splenic AA amyloid and its presence accelerates SAA fibril formation. In addition, Asc-/- bone marrow-derived macrophages did no longer exhibit increased SAA-stimulated phagocytosis activity in vitro, suggesting an altered macrophage processing in the absence of ASC.
In summary, we conclude that ASC modulates both SAA serum levels and the severity of AA amyloidosis in mice and may, therefore, be a potential therapeutic target in AA amyloidosis.
Results
Amyloid induction
To investigate the role of ASC in the pathogenesis of AA amyloidosis in vivo, Asc wt and Asc-/- female and male F2 generation littermates were injected with AgNO3 and preformed AA fibrils (amyloid-enhancing factor, AEF) for AA amyloidosis induction (AA mice), whereas control mice were only injected with PBS, AEF or AgNO3 (Fig. 1a-b). 79 mice were included in this study and AA was induced in 22 Asc wt and 24 Asc-/- mice. A total of 33 mice were only injected with either PBS, AgNO3 or AEF and served as controls (Table S1). The experimental overview including injection schematic, blood withdrawals and euthanasia regimen is illustrated in Fig. 1b. To assess AA amyloid deposition over time, mice were sacrificed either at day 16 or at day 30, two days after the last AgNO3 injection. The presence of amyloid in spleens was assessed by staining histological sections of paraffin-embedded tissue with Congo red, upon which we saw the characteristic apple-green birefringence under polarized light (Fig. S1). No amyloid could be detected in control group mice that were only injected with either PBS, AgNO3 or AEF. The detailed characteristics of the experimental groups are summarized in Table S1. Age distribution did not differ significantly between experimental groups (Table S2).
The recruitment of Serum Amyloid A in AA amyloidosis is modulated by ASC
The development of AA amyloidosis is a consequence of excessive production and recruitment of SAA. We, therefore, assessed SAA serum levels in Asc wt and Asc-/- mice by enzyme-linked immunosorbent assay (ELISA) at baseline (before any injection) as well as at 9, 24, 48, 72- and 96-hours post injection (hpi) of AgNO3 and AEF (Fig. 1b and Fig. 2). At baseline and at 9 hpi, there was no difference between mean SAA serum concentrations of all experimental Asc wt and Asc-/- groups. However, at 24 hpi, the timepoint at which we expected serum acute-phase proteins to peak (1, 37), we observed differences in SAA serum levels between experimental groups, depending on Asc genotype and the presence or absence of AEF. SAA serum levels of Asc wt mice only treated with AgNO3 were significantly higher than in Asc wt mice with AgNO3+AEF (p=0.0348), suggesting that SAA is recruited in the presence of preformed AA fibrils and therefore decreased in serum. Furthermore, the decrease in SAA serum concentration observed in Asc wt AA mice, was abrogated in Asc-/- AA mice (p=0.0035). Mean SAA serum concentrations of Asc-/- AA mice were slightly higher than the SAA serum concentration of the AgNO3-only injected Asc-/- groups at 9, 24, 48 and 72 hpi (p=0.0604 at 72 hpi), possibly because the ELISA detected injected preformed AA fibrils that were still present in the blood stream (Fig. S2).
As presumed, 48 hours after injection, SAA serum concentrations decreased in all experimental groups. However, at this time point the difference between Asc wt and Asc-/- AA mice was still significant (p=0.0048). Depending on the genotype, the difference between SAA serum concentrations of the two Asc wt and Asc-/- AA induced groups persisted up to 72 hours after injections (p=0.034).
Finally, 96 hours after injection there was no longer any significant difference in SAA serum concentration. Overall, Asc-/- AA mice were found to have the highest SAA serum concentration among all experimental groups. Based on these results, we postulate that in experimental amyloidosis, ASC facilitates the recruitment and deposition of SAA in murine AA amyloidosis. Detailed SAA serum values are given in Table S3.
AgNO3-induced acute-phase responses in Asc-/- and wildtype mice
While it is known that aluminum-containing adjuvants activate the NLRP3 inflammasome (38), it is not known whether AgNO3 stimulates ASC-dependent inflammasome pathways of innate immune cells. To investigate this question, we assessed SAA levels of AgNO3-only injected Asc wt and Asc-/- mice by ELISA and compared their SAA serum concentrations at baseline and up to 96 hours after injection (Fig. 1b and Fig. 2).
As mentioned previously serum concentrations of SAA and other acute-phase proteins peak at around 24 hours after a proinflammatory stimulus. At this time, Asc genotype did not significantly impact SAA serum levels after AgNO3 stimulation, as there was no significant difference in SAA serum concentration between Asc wt and Asc-/- mice that had only been treated with AgNO3. The same was also true for all successive timepoints. Genetic ablation of Asc did not significantly impact AgNO3-induced SAA levels, suggesting that ASC-dependent inflammasomes do not significantly contribute to AgNO3 sensing. Given that the experimental AA amyloidosis model requires an AgNO3 treatment to induce inflammation, we conclude that applying the murine AA amyloidosis model to Asc-/- mice would allow us to study the role of ASC in SAA recruitment and AA amyloid formation.
Decreased splenic amyloid deposition in the absence of ASC-dependent inflammasomes
In mice with experimental AA amyloidosis, we observed that amyloid deposition commenced in the marginal zone of the white pulp, followed by red pulp invasion, which is in accordance with other studies (31–33). To assess amyloid load, we stained spleen sections with the luminescent conjugated polythiophene (LCP) HS-310. Representative spleen sections of Asc wt and Asc-/- AA mice are shown in Fig. 3a-d. Overall, Asc wt mice exhibited more pronounced red pulp invasion of amyloid compared to Asc-/- mice (white arrows). In the PBS-only, AEF-only and AgNO3-only treated control mice, no amyloid could be detected (Fig. S1 and Fig. S3). To quantify the amyloid load of AA mice, we calculated, based on fluorescence, the integrated density, which is the sum of the values of the pixels in the region of interest and the percentage of the amyloid area deposited within spleen sections (HS-310+). The mean fluorescence integrated density (A.U./µm2) differed significantly between Asc wt AA mice of the 16-days and the 30-days group (p=0.0035), whereas it did not in Asc-/- AA mice (Fig. 3e). In addition, there was a significant difference in amyloid load between Asc wt and Asc-/- mice of the 30-days group (p=0.0242). Similar results were seen in the analysis of the amyloid area deposited in the spleen. There was a significant increase in amyloid from day 16 to day 30 in Asc wt AA mice (p=0.0004), whereas the increase of amyloid Asc-/- AA mice was attenuated and not significant within the same period. Furthermore, Asc-/- AA mice from the 30-days group revealed a decreased amyloid deposition compared to Asc wt AA mice (p=0.0125) (Fig. 3f). Detailed area values are given in Table S4. Using Western Blot (WB), we assessed the presence and quantity of SAA in spleen homogenate of four Asc wt and five Asc-/- AA mice with the highest splenic amyloid load, as assessed by LCP staining (Fig. 3g-h). In WB we detected bands with sizes below 12 kDa, which probably represented proteolytic degradation products of AA amyloid (39). Moreover, ASC was found to be highly expressed, adjacent and integrated to amyloid deposits throughout the spleen of Asc wt AA mice (Fig. 4a). In summary, these data suggest that AA amyloid load and deposition depend on Asc genotype.
Accelerated SAA fibrillation in presence of ASC
In order to assess the effect of ASC on SAA fibril formation we performed an in vitro SAA aggregation assay in presence as well as in absence of ASC specks (Fig. 4b). The halftime (t1/2), where the relative aggregation concentration is 0.5, was significantly reduced in presence of ASC specks and inversely correlated with increased concentrations. The t1/2 of murine SAA aggregation was significantly reduced in presence of 100k ASC specks compared to the samples that only contained murine SAA (p=0.0282). Furthermore, in presence of 200k and 500k ASC specks the lag phase was further reduced significantly, respectively (p=0.0075, p<0.0001) (Fig. 4c). Hence, this suggests that the presence of the ASC protein accelerates SAA fibril formation. Detailed values are given in (Table S5).
Splenic architecture and peripheral blood in AA amyloidosis
Prolonged elevation of serum SAA is required to trigger AA amyloidosis (5). Once present, AA aggregates progressively disrupt tissue integrity and ultimately impair physiological function of affected organs. Furthermore, patients with systemic AA amyloidosis may also exhibit altered red blood cell and platelet volumes according to recent reports (40, 41). Moreover, the presence of platelets in the circulation and platelet-derived enhancing factors supply inflammasome activation and boost inflammasome capacity of macrophages, neutrophils and monocytes in inflammation (42). Since SAA activates the NLRP3 inflammasome of innate immune cells, as described above, we assessed the cellular composition of the spleen and of peripheral blood at baseline and after induction of experimental AA amyloidosis, in Asc wt and Asc-/- mice. We assessed B cells, T cells, dendritic cells, neutrophils as well as macrophages including M1- and M2-like macrophages in the spleen (Fig. S4). Altogether, mice that had only been treated with AgNO3 as well as mice with AA induction had an increased splenic macrophage infiltration compared to baseline state, underscoring the involvement of innate immune cells in AA amyloidosis. Macrophage infiltration was neither dominated by M1- nor by M2-like macrophages (Fig. S4c). Furthermore, we assessed the transcriptional state of splenic macrophages from Asc wt and Asc-/- AA mice by RNA sequencing (Fig. S5). Significant (p<0.05 and fdr<0.01) transcriptional changes between specimen were mainly found in the Asc gene (Fig. S5c and Fig. S5d). Lymphocyte, monocyte, granulocyte, red blood cell and platelet counts were additionally assessed in peripheral blood (Fig. S7 and Fig. S8). Interestingly, platelet count at baseline was higher in Asc-/- compared to Asc wt mice (p=0.0172). The same accounted for Asc-/- AA mice from the 30-days group (p=0.0357) (Fig. S7).
Impaired phagocytic activity of SAA-activated Asc-/- bone marrow-derived macrophages
Macrophages and monocytes play a central role in AA amyloidosis. First, monocytes are sufficient to transfer AA amyloidosis in vivo (43). Furthermore, macrophages co-localize with AA amyloid in the spleen of AA mice (32). Moreover, phagocyte depletion delays or inhibits AA amyloid accumulation (31, 35). Another important fact is that AA amyloid undergoes Fc-receptor-mediated phagocytosis by macrophages, which is initiated by host-specific antibodies that target AA protein (36). Given the importance of monocytes and macrophages in AA amyloidosis, that SAA activates macrophages (29, 30) and that Asc heterozygous astrocytes have an increased Aβ phagocytosis in AD mice (44), we sought to test whether ASC may modulate the phagocytic activity of Asc wt and Asc-/- murine BMDMs in vitro (Fig. S6a-b). We, therefore, exposed SAA-activated and non-(SAA)-activated murine BMDMs to an in vitro phagocytosis assay (Fig. S6c). There was no significant difference in phagocytic activity between unstimulated BMDMs of both Asc genotypes. The highest phagocytic activity among the murine BMDMs was seen in SAA-activated Asc wt BMDMs as compared to non-stimulated Asc wt BMDMs (p=0.0015). Compared to their wildtype counterparts, we observed lower phagocytic activity in SAA-activated Asc-/- BMDMs. Furthermore, there was a difference in phagocytic activity between SAA-stimulated Asc wt and Asc-/- BMDMs (p=0.0002). Hence, the Asc genotype indeed affects the phagocytosis of SAA-activated BMDMs. Nevertheless, the absence of SAA-induced phagocytosis stimulation in Asc-/- BMDMs (compared to Asc wt BMDMs) supports the fact that the ASC protein is crucial for the decreased AA amyloid deposition in Asc-/- mice rather than the altered phagocytosis in absence of ASC per se. Detailed values are given in (Table S6).
Discussion
Protein misfolding diseases and their associated degenerative disorders display a major socioeconomic burden worldwide and scarcity of specific therapies still prevails.
In the present study we investigated the role of ASC in the pathogenesis of systemic AA amyloidosis in vivo. To do so, we harnessed an experimental platform of AA amyloidosis and employed mice in which Asc, the gene coding for the central component of the NLRP3 inflammasome, had been ablated (45). We report that ASC modulates SAA serum levels and controls the severity of murine AA amyloidosis. Given its potential to influence amyloid formation, ASC may represent a valid therapeutic target for the treatment of AA amyloidosis. A fundamental role of the innate immune system is the sensing of environmental danger signals, which is mediated by the family of cytosolic pattern recognition receptors (PRR). Nalp3, the receptor of the NLRP3 inflammasome, is a PRR that mediates the immunostimulatory properties of aluminum containing adjuvants (38). Therefore, we began by assessing whether silver nitrate (AgNO3), the proinflammatory stimulus required to trigger murine AA amyloidosis in this study, is sensed by ASC-dependent inflammasomes and whether AgNO3 injection leads to a similar increase in SAA serum concentrations in Asc-/- and Asc wt mice. AgNO3-only injected Asc-/- mice showed mean SAA serum elevations that were comparable to those in Asc wt mice that had received the same treatment. We conclude that ASC inflammasomes do not play a major role in silver nitrate sensing and that the murine AA amyloidosis model was suitable for our in vivo study in Asc-/- animals.
In order to determine the role of ASC in SAA kinetics upon an inflammatory stimulus, we assessed the temporal development of SAA serum concentrations of Asc wt and Asc-/- experimental mice. Peak SAA concentration was reached 24 hours after injection; at this timepoint SAA serum levels of Asc wt mice with experimental AA amyloidosis were significantly decreased, compared to Asc wt mice that had solely been treated with AgNO3 (no AEF). Intriguingly, in the presence of injected preformed AA fibrils (AEF) but absence of endogenous ASC, serum SAA concentrations of Asc-/- mice remained increased up to 72 hours post injections. These data suggest that ASC may serve as an enhancing factor for the aggregation of SAA and AA templated by preformed AA fibrils. SAA levels only decreased in the presence of ASC, suggesting that ASC may mediate a SAA serum-to-tissue or serum-to-cell translocation.
We then checked for the presence of amyloid after AA induction using Congo red and LCP stains in all experimental groups. We found that only mice who had undergone the full experimental AA amyloidosis induction protocol with AgNO3 and AEF injections, but none of the control mice, showed amyloid deposition.
ASC speck co-localization with Aβ plaques occurs in AD brains (25). Furthermore, there is evidence that ASC co-localizes with AA amyloid in kidney biopsies from FMF patients suffering from AA amyloidosis (28). If ASC has a propensity to bind amyloids generically, it may form complexes with AA amyloid as well. Indeed, we detected the presence of ASC within AA amyloid deposits in spleen tissue from Asc wt AA mice. In presence of ASC, the deposition of AA amyloid appeared to be more invasive into the red pulp than Asc-/- AA mice, where amyloid was mainly found in proximity to vessels and limited to the marginal zones of follicles. Furthermore, the presence of ASC specks facilitated and accelerated SAA fibril formation in vitro, which supports the idea that ASC inflammasome protein assemblies serve as a generic protein aggregation-enhancing platform in various central and peripheral PMDs where the innate immune system is involved. It has recently been reported that there are few ASC specks in fully matured lymphoid organs, which might explain their rather sparse distribution in the examined tissues (46). However, amyloid was significantly reduced in Asc-/- compared to Asc wt mice after 30 days of inflammation. AA amyloid consists of multiple components such as serum amyloid P, proteoglycans, glycosaminoglycans, lipid rafts as well as SAA (47, 48). Although SAA displayed a non-significant difference in western blot analysis, the ASC protein incorporation into amyloid of Asc wt mice with AA as well as the compelling amyloid load reduction in Asc-ablated mice with AA (as visible in LCP-stained tissue) strongly advocate ASC as the driving force in the pathogenesis of AA amyloidosis. SAA western blot analysis of spleen homogenate from AA mice revealed multiple bands running between 8 and 12 kDa in size. Murine SAA has a mass of approximately 11.8 kDa. The bands <12 kDa in size most likely represent proteolytic degradation products of AA amyloid (39, 49).
It is widely recognized that macrophages co-localize with amyloid deposits and that Fc-receptor-mediated phagocytosis plays an important role in deposition, processing and clearing of AA amyloid (31, 32, 36, 39, 50). We, therefore, tested another experimental paradigm, where we aimed to elucidate the role of ASC in the phagocytic activity of macrophages. Interestingly, Asc+/-, but not Asc-/-, astrocytes exhibit increased phagocytosis of Aβ in a mouse model of AD (44). Moreover, macrophages and monocytes are capable of converting endocytosed SAA to processed extracellular AA amyloid in vitro (51, 52). We, therefore, investigated whether Asc-/- macrophages reveal enhanced phagocytic activity, as this might be contributory to altered and less AA amyloid deposition or AA processing. We observed that SAA-stimulated Asc wt murine BMDMs showed increased phagocytosis, compared to Asc-/- macrophages. These findings suggest that the reduced splenic AA amyloid in Asc-/- mice load is mainly due to the absence of ASC and not due to enhanced phagocytosis of Asc-/- BMDMs. In addition, transcriptomic analysis of splenic macrophages of AA diseased mice revealed mainly Asc gene upregulation among wildtype specimen. These data further underline the crucial role of ASC in AA amyloidosis. Nonetheless, an impaired phagocytosis of SAA-activated Asc-/- macrophages might potentially contribute to altered AA processing and removal.
Finally, we investigated whether splenic and peripheral blood cellular alterations occurred in Asc wt and Asc-/- AA mice. We assessed splenic cellular composition with a focus on B cells, T cells, dendritic cells, neutrophils and macrophages (including M1- and M2-like polarized macrophages), together with peripheral blood composition of lymphocytes, monocytes, granulocytes, red blood cells and platelets. There was an increased splenic macrophage infiltration in AgNO3-only and AA induced mice at day 16 and 30, compared to baseline levels, which underscores the involvement of innate immune cells in the pathogenesis of AA amyloidosis.
When comparing peripheral blood composition of Asc wt to Asc-/- mice, we observed a slightly increased baseline platelet count of Asc-/- mice as well as marginally increased platelets numbers in Asc-/- AA mice at day 30 in peripheral blood. According to a recent report, platelets release enhancing factors that boost the inflammasome activation of innate immune cells (42). We speculate that in the absence of ASC, platelets might be increased as part of a compensatory mechanism.
In summary, we conclude that ASC modulates SAA serum levels, accelerates SAA fibril formation and controls the severity of AA amyloidosis. Although we have identified ASC as a critical protein in the pathogenesis of AA amyloidosis, additional work may be required to resolve the precise molecular mechanism of its involvement. It is conceivable that the modulatory effect is not only a result of the binding properties of ASC and ASC specks for β-sheet rich proteins. However, the findings of this study highlight a crucial role of ASC in SAA recruitment and amyloid deposition in AA amyloidosis, which might also be relevant in the pathogenesis of further systemic amyloidoses. It may very well be that ASC and its protein assemblies serves as aggregation enhancing platform in various PMDs. Finally, it is possible that our findings might have therapeutic implications, since ASC could be a target of disease-modifying therapies that aim to reduce amyloid deposition in various PMDs.
Material and Methods
Mice
Animal care and experimental protocols were in accordance with the Swiss Animal Protection Law and approved by the Veterinary office of the Canton of Zurich (permit ZH131-16). Mice were bred in a high hygienic grade facility of the University Hospital of Zurich (BZL) and housed in groups of 2-5. Mice were under a 12 hours light/ 12 hours dark cycle (from 7 a.m. to 7 p.m.) at 21 ± 1°C. Asc-deficient mice (B6.129-Pycardtm1Vmd) were generated as previously published in (45). C57BL/6 wildtype mice were obtained from the Jackson laboratory. To minimize environmental bias and potential differences in microbiota, F2 littermates were bred. We perfomed the experiments with highest possible gender and age congruence among experimental groups (Table S1-S2). Mice were randomly assigned to the experiments.
Genotype screening
Ear biopsies were digestd and subjected to PCR. An 859 bp Asc allele fragment was amplified using forward 5’-GAAGCTGCTGACAGTGCAAC-3’ and reverse 5’-CTCCAGGTCCATCACCAAGT-3’ primers. Amplification of a 275 bp gDNA fragment from the B6.129-Pycardtm1Vmd Neo cassette was done using forward 5’-TGGGACCAACAGACAATCGG-3’ and reverse 5’-TGGATACTTTCTCGGCAGGAGC-3’ primers. PCR products were run on a 1.5% agarose gel and developed for genotype definition.
AA induction, injections and reagents
Silver nitrate (Merck) was eluted in nuclease-free water (Ambion®). AEF was prepared as previously described in (53) and pH was adjusted to 7.4 before administration. AA amyloidosis induction performed by injections of 100 µl AEF (i.v.) and 200 µl sterile-filtered 1 % silver nitrate solution (s.c.). Repeated injections of silver nitrate were in accordance to (39) (Fig. 1).
Mouse serum amyloid A (SAA) measurements
To determine the SAA levels in mice serum, blood was withdrawn into BD Microtainer® SSTTM Tubes at baseline and up to 96 hours post injection. Samples were left at room temperature (RT) for 30 min and subjected to centrifugation at 10’000xg for 8 min at 4°C. Sample sera were then transferred and stored at −80°C. SAA levels were assessed by a mouse SAA enzyme-linked immunosorbent assay (ELISA) kit (abcam) according the manufacturers’ guidelines. Mouse serum samples were analyzed in technical triplicates. ELISA plate was developed using of 450 nm for absorbance.
Euthanasia and organ harvesting
Upon euthanasia, organs were harvested and kept on ice in Iscove’s Modified Dulbecco’s Medium (IMDM) (ThermoFisher Scientific) until measurement and further usage (no longer than 4 hours). Bone marrow cells of tibia, femur and pelvis were flushed into IMDM using 25G needles (B. Braun). Tissue was fixated in formalin for paraffin embedding or put in Tissue-Tek® O.C.T.TM compound (Sakura®) for frozen sections.
Histology and immunohistochemistry
Formalin-fixed and paraffin-embedded spleen sections (2-4 µm) were de-paraffinized with three cycles of xylene treatment followed by re-hydration with 100 % EtOH, 96 % EtOH, 70 % EtOH and water (each cycle 5 min) respectively. Hematoxylin and eosin (HE) as well as Congo red (CR) staining and ASC immunohistochemistry (IHC) were performed according to standard procedures used at our institute. IHC was performed with anti-ASC pAb (clone AL177, AdipoGen) at a dilution of 1:500. Slides were incubated with HS-310 for 30 min at RT in the dark at a final concentration of 0.3 µg/ml in PBS (54, 55). After washing, slides were mounted with fluorescence mounting medium (Dako) and proceeded to fluorescence microscopy for imaging (OLYMPUS BX61 fluorescence microscope system harnessing an OLYMPUS XM10 camera). The hexameric LCP HS-310 was produced as previously described (56).
Fluorescence and polarized microscopy
To analyze LCP-stained tissue sections, we assessed three different and independent visual fields at 4x magnification and slides per mouse and organ. Two parameters, HS-310 positive area (% of area) as well as the fluorescence integrated density (A.U./µm2) of HS-310 were calculated with the free software application ImageJ (imagej.net). To confirm the presence of amyloid, we assessed the apple-green birefringence of amyloid under polarizing light in Congo red stained spleen tissue sections (Fig. S2). FACS sorted splenic macrophages were visually confirmed by filter settings that allowed the detection of PE-Cy5 and APC-Cy7.
Immunoblot analysis
To determine SAA presence by WB, spleen tissues from AA induced mice were homogenized in 1:9 volumes (w/v) of RIPA buffer (50 mM Tris pH 7.4, 1 % NP-40, 0.25% Deoxycholic acid sodium salt, 150 nM NaCl, 1 mM EGTA, protease inhibitors (complete Mini, Roche)) using TissueLyser LT for 45 seconds for four cycles. Samples were cooled on ice between cycles. Supernatant was transferred into new tubes after full speed centrifugation for 10 min at 4°C. 10 µl spleen homogenate was boiled at 95°C for 10 min with a final concentration of 1 µM DTT and 4x NuPageTM LDS sample buffer (ThermoFisher Scientific). Spleen homogenate was separated using SDS-PAGE (Novex NuPAGE 4-12 % Bis-Tris Gels, Invitrogen) and transferred to a PVDF membrane (ThermoFisher Scientific) at 20 V for 7 min. Membrane was blocked with 5% milk in TBS-T (Tris-Buffered Saline, 0.1 % TWEEN®20, pH 7.6) for 3 hours at RT. Primary rabbit anti-SAA antibody was overnight (o/n) incubated at 4°C at a concentration of 2.5 µg/ml (Table S7). After 4 cycles of washing, membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Jackson Immuno) diluted 1:5000 in blocking buffer for 1 hour at RT. Blots were developed harnessing Luminata Crescendo Western HRP substrate (Milipore) and visualized with the Stella system (model 3200, Raytest). Actin served as loading control. Membranes were sequentially incubated with primary mouse anti-actin antibody (Merck) at 1:8000 in blocking buffer at 4°C o/n. After washing membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (Jackson Immuno) diluted 1:8000 in blocking buffer for 1 hour at RT. Blots were developed as described above (Fig. S9). WB quantification was performed using the free software Image StudioTM Lite (licor.com).
In vitro SAA fibril formation
Recombinant murine SAA1 was generated as previously described in (57). The in vitro aggregation assay was carried out in a black 96-well plate (Greiner Bio-One, PS, F-bottom, black) on a FLUOstar OMEGA plate reader (BMG Labtech). SAA1 was dissolved in water at a stock solution concentration of 10 mg/ml. The final reaction volume was 100 µl per well and consisted of 50 µM murine SAA1, 20 µM Thioflavin T (abcam) and 10 mM Tris buffer pH 8.0. ASC specks were recombinantly produced as previously described (58), diluted in PBS and added at various concentrations. The plate was agitated every 20 min by orbital shaking for 10 s at 100 rpm. The assay was performed at 37°C. Fluorescence (Ex: 450nm, Em: 490 nm) was measured over the course of 140 hours. Data was normalized using the free software application AmyloFit according to the developers’ guidelines (59).
Differentiation of bone marrow cells to bone marrow derived macrophages (BMDMs)
BMDMs were generated, with slight modifications, as previously published (60). BM cells were harvested from tibia and femur, washed twice in PBS and resuspended in differentiation medium I-10 + M-CSF (i.e. IMDM, 10 % FBS, 1 mg/ml Pen/Strep containing 25 ng/ml recombinant murine M-CSF (PeproTech)) at a density of 1-2 Mio. cells/ ml. After incubation for four days at 37°C differentiation medium was exchanged. On day 8 attached BMDMs were detached using Accutase® (Innovative Cell Technologies, Inc.), washed twice in PBS, counted and proceeded to the phagocytosis assay.
In vitro phagocytosis
The phagocytosis of BMDMs was performed using a phagocytosis assay kit (abcam). Individual samples were prepared in duplicates. The in vitro assay was performed in 96-well tissue culture plates (TPP). Asc wt and Asc-/- BMDMs were suspended at a density of 500’000 cells/ml. BMDMs were stimulated, with slight modifications, as previously published (30). BMDMs were stimulated with murine SAA1 at a concentration of 2 µg/ml for 16 hours at 37°C prior to phagocytosis substrate encounter. After 1-hour phagocytosis substrate incubation at 37°C, cells were treated according to the manufacturers’ guidelines and proceeded to EnVision Multimode Plate Reader (PerkinElmer) for data acquisition. The absorbance was determined at 405 nm.
Assessment of cellular spleen architecture by flow cytometry
After sacrifice, cells were isolated from spleens using a 70 µm cell strainer (Falcon®). Following red blood cell lysis cells were stained and with the following monoclonal antibodies to determine cellular architecture of most abundant spleen and immune cells (Table S8): anti-mouse CD45.2 (Biolegend), anti-mouse Gr-1 (eBioscience), anti-mouse F4/80 (eBioscience), anti-mouse CD11b (Biolgegend), anti-mouse B220 (Biolegend), anti-mouse CD3 (Biolgend), anti-mouse CD206 (Biolegend), anti-mouse MHCII (Biolegend) and anti-mouse CD11b (eBioscience). Data acquisition and analysis was performed on a BD LSRIIFortessaTM flow cytometer and FlowJo v10.6.1, respectively. Gating strategy was applied, with slight modifications, as previously published (61–63).
RNA extraction and high throughput sequencing (NGS)
Upon FACS sorting of F4/80+/CD11b+ splenic macrophages originating from AA mice (see above) total RNA was extracted using TRIzolTM Reagent (ThermoFisher Scientific) according to the manufacturers’ guidelines and proceeded to high throughput sequencing (NGS). RNA integrity and quantity were assessed by RNA ScreenTape Analysis (Agilent). The SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian (Takara) together with a NovaSeq platform (Illumina) was applied for transcriptomic data acquisition. Data was analyzed using established analysis pipelines at the Functional Genomics Center Zurich (FGCZ).
Assessment of complete blood count (CBC) from peripheral blood
Blood was withdrawn into Microvette® 100 K3E (SARSTEDT Germany) cuvettes according to the manufacturers’ guidelines. To assess CBCs, samples were run on an ADVIA (Siemens Healthineers) hematology system.
Statistical analysis
Statistical analysis was performed using Graph Pad Prism v8.0.0. If not indicated otherwise, unpaired, two-tailed Student’s t-tests or one-/two-way ANOVA were performed, followed by post hoc analysis wherever appropriate. Statistical details are described in the respective figures. A two-tailed p-value <0.05 was considered as statistically significant. Confidence intervals were calculated at a confidence level of 95 %.
Statement of author contribution
Conceived and designed the experiments: ML, AA. Performed the experiments: ML, PS, VL, ICM, MC, MD. Analyzed the data: ML, PS. Contributed reagents and materials: KF, AKKL, GTW, KPRN, MN, SH. Wrote the manuscript: ML. Edited manuscript: all authors.
Declaration of interests
The authors declare no competing interests.
Supplemental figures
Supplemental tables
Supplemental material and methods tables
Acknowledgments
We thank Rita Moos, Paulina Pawlak and Prof. Dr. Michael Heneka for advice and expertise. We appreciate that Dr. Emmanuel Contassot shared the B6.129-Pycardtm1Vmd mice. We thank Dr. Marcus Fändrich and his colleagues for providing murine SAA1 protein. A.P.A.T. is supported by the Professor Dr. Max Cloëtta Foundation. AA is the recipient of an Advanced Grant of the European Research Council, the Swiss National Foundation, and the Nomis Foundation.