The ASC inflammasome adapter controls the extent of peripheral protein aggregate deposition in inflammation-associated amyloidosis
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Abstract
ASC-containing inflammasomes form specks, extracellular aggregates which enhance the aggregation of Aβ amyloid in Alzheimer’s disease. This raises the question whether ASC participates to additional aggregation proteinopathies. Here we show that ASC controls the extent of inflammation-associated AA amyloidosis, a systemic disease caused by the aggregation of the acute-phase reactant serum amyloid A (SAA). Using superresolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Purified recombinant ASC specks accelerated SAA fibril formation in vitro . Mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain. In a murine model of inflammation-associated AA amyloidosis, splenic AA amyloid load was conspicuously decreased in Pycard tm1Vmd/tm1Vmd mice which lack ASC. This reduction was not a consequence of enhanced amyloid phagocytosis, as SAA stimulation increased phagocytic activity in Pycard +/+ , but not in Pycard -/- macrophages. Treatment with anti-ASC antibodies decreased the amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (-logEC 50 ≥ 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. Higher anti-ASC titers did not correlate with any specific disease, suggesting that anti-ASC immunotherapy may be well-tolerated. These findings expand the role played by ASC to extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.
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Reply to the reviewers
Manuscript number: RC-2023-02235
Corresponding author: Adriano, Aguzzi
1. General Statements
We thank the reviewers for providing valuable comments. We are pleased that our study is considered important to advance the knowledge on IL-1-independent inflammatory functions of inflammasomes. We have clarified and revised the manuscript (track changed) as detailed below in the point-by-point response in this letter.
2. Point-by-point description of the revisions
Referee 1
General: In this manuscript, et al., investigates the role of the inflammasome adapter ASC (in AA amyloidosis). This condition involves the aggregation of serum amyloid A (SAA) and is …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Manuscript number: RC-2023-02235
Corresponding author: Adriano, Aguzzi
1. General Statements
We thank the reviewers for providing valuable comments. We are pleased that our study is considered important to advance the knowledge on IL-1-independent inflammatory functions of inflammasomes. We have clarified and revised the manuscript (track changed) as detailed below in the point-by-point response in this letter.
2. Point-by-point description of the revisions
Referee 1
General: In this manuscript, et al., investigates the role of the inflammasome adapter ASC (in AA amyloidosis). This condition involves the aggregation of serum amyloid A (SAA) and is linked to chronic inflammation. Firstly, I can directly say that I do recommend this study for publication. This is a well conducted and well-written study which advances the knowledge on IL-1-independent inflammatory functions of inflammasomes. Furthermore, I find it particularly impressive that despite the inflammasome research community is well aware that amyloidosis is a hallmark of inflammatory diseases, it took a neuroscientist specialized in prion diseases to raise the question whether ASC would be involved in seeding serum AA aggregation. Key findings include: • ASC forms extracellular aggregates that enhance SAA aggregation, as observed through superresolution microscopy. • In a mouse model, the absence of ASC significantly reduced amyloid load, not due to increased phagocytosis but likely due to diminished aggregation. • Treatment with anti-ASC antibodies reduced amyloid load and mitigated weight loss in mice with AA amyloidosis. These findings suggest that ASC plays a crucial role in AA amyloidosis and that targeting ASC could be a potential therapeutic strategy. The study expands our understanding of the involvement of ASC in proteinopathies beyond neural diseases, pointing to its role in systemic conditions like AA amyloidosis.
__*Significance: *__In conclusion, this manuscript offers valuable insights into the role of ASC in AA amyloidosis, presenting compelling findings that support its potential as a therapeutic target. Addressing the mentioned concerns and making the suggested revisions will further enhance the manuscript's scientific rigor and impact. Overall, this study is a valuable contribution to the field of inflammasome research and its relevance in systemic conditions like AA amyloidosis.
* *
Comment 1: Overall, the experiments are well-conducted and mostly all controls I would expect were included. With few exceptions, the data is convincing. With that said, I have issues with some of the staining employed in Fig 1. In Fig. 1, the authors assess ASC staining in cardiac tissues from a patient with vasculitis and systemic inflammation-related AA amyloidosis, and a control patient who died of a heart attack but had no signs of amyloidosis. However, most of the data shown is related to the AL177 anti-ASC. More importantly, no isotype stainings are included. We have previously demonstrated that the AL177 anti-ASC, used here, reacts quite strongly with ASC−/− cells, and it is one of the less specific anti-ASC commercially available (PMID: 27221487). As this is data from one patient (understandably), I wonder if the authors could counterstain ASC in the same samples using a specific human anti-ASC with a different color (ex: Biolegend HASC), and confirm that the signal overlays with the AL-177.
Response: We conducted additional experiments to address the anti-ASC antibody specificity, as now described in Results, Method, and Fig. S1. We tested a set of anti-ASC antibodies (AL177, MY6745, 1C3D7) for their ASC specificity. We confirmed that both the AL177 and the MY6745 antibodies have high ASC-specificity (Fig. S1A). Moreover, for illustration purposes (and to warn other scientists), we included a third anti-ASC antibody (1C3D7) found to be unspecific as it yielded a strong signal in PYCARD-/- (ASC-/-) THP-1 cells (Fig. S1B). In addition, isotype controls were included in these experiments (Fig. S1A, right panels), as suggested by the reviewer, showing no target protein detection in both, PYCARD+/+ (ASC+/+) and PYCARD-/- cells underscoring the anti-ASC specificity of AL177 and MY6745 antibodies.
Comment 2: Finally, in Figure 1H it seens from the description that another anti-ASC was used: "referred in the legend as ASC (MAB ASC, Yellow)". Is this a monoclonal anti-ASC? Also, the images show large and bright antibody aggregates (middle of the image, top left corner behind the "H", and a massive fluorescence in the bottom right of the image), indicating the presence of staining artifacts. Again, no counterstaining with isotype controls are shown.
Response: We apologize for the confusing jargon in Figure 1H. “MAB ASC” refers to the anti-ASCPYD antibody (MAB/MY6745). We have corrected the antibody terminology in the legend. MAB/MY6745 is a monoclonal antibody generated by Mabylon that is highly reactive to both human and murine ASC. This antibody was generated to 1) perform an immunotherapy in vivo study and to 2) be used as alternative specific antibody in addition to AL177 to show co-localization of SAA and ASC in a human AA patient using STED superresolution microscopy. MAB/MY6745 is a rabbit monoclonal anti-ASC antibody targeting the pyrin domain (PYD) from which the rabbit Fcγ domain was replaced with that of a mouse IgG2a domain to avoid xenogeneic anti-drug responses in recipients and to improve its effector functions in vivo. To examine possible staining artefacts which can occur with Formalin-Fixed Paraffin-Embedded (FFPE) human tissues, we assessed the specificity of a variety of anti-ASC antibodies (Fig. S1). Our data presented in Fig. S1 show that the monoclonal anti-ASC antibody binds specifically. It is conceivable that AL177 and MAB/MY6745 target different epitopes of ASC, resulting in different staining patterns. An isotype control, included in __Fig. S1, __was used to test the specificity of the secondary antibodies, and did not show any nonspecific staining. We have adapted and added this to the text body and figure legend accordingly.
Comment 3: Overall, although I don't dispute the possibility that ASC would co-localize with SAA deposits, I don't think the data presented can safely sustain that claim. I would, therefore, suggest that alternative methods to be employed to substantiate these conclusions: Supposedly, would it be possible to immuno-precipitate (IP) amyloid SAA and assess ASC via western blotting? As well as IP ASC and detect SAA? Or use DSS-crosslinking to find ASC oligomers in tissue areas rich in SAA?
Response: In addition to assessing co-localization by means of STED superresolution microscopy (Fig. 1), we also employed LiP-MS with various forms of ASC (monomeric and ASC specks) and identified a previously unrecognized biophysical interaction of SAA and the ASC PYD domain (Fig. 2C-F). As an orthogonal line of evidence, we provided kinetic data showing that SAA aggregation is enhanced in the presence of ASC specks (Fig. 2A-B). We feel that these results are reasonably convincing, but we agree that co-localization is almost invariably an aspirational finding, and even superresolution microscopy cannot fully exclude the presence artifacts (nor can, in fairness, co-immunoprecipitation, which must often rely on overexpression). A sentence acknowledging this limitation was added to the Discussion.
Comment 4: For example, it would be reasonable to quantify the results in Figure 3G and providing clarification regarding the controls in the figure legend. Though there is significantly less SAA in spleen homogenates from Asc−/−, there also seems to be the case for b-actin in Fig 3G. Moreover, in the figure legend the authors state: "...Spleen homogenate from untreated (-ctrl) and AA+ (+ctrl) C57BL/6 wt mice from an independent experiment served as negative and positive control, respectively." I don't know what the authors mean with that. Is this a montage, or samples from different experiments were run together in one blot? And if so, for what reason? This is confusing and should be clarified.
Response: We reworded the figure legend to provide clarity about the technical assay controls and adjusted the labels in __Fig. 3E __accordingly: To ascertain SAA antibody functionality, mouse spleen homogenate from independently obtained and Congo red-confirmed AA+ tissue served as positive, whereas non-induced (AA-) spleen tissue served as negative technical controls. (Fig 3E). We decided to show the two (positive/AA+ and negative/AA-) technical controls in Fig. 3E.
Comment 5: Furthermore, in the Abstract, a slight rephrasing is suggested to accurately describe ASC specks as molecular aggregates formed inside cells, which are subsequently released into the extracellular space.
Response: We thank the referee for bringing this to our attention. We rephrased the abstract accordingly.
Comment 6: Lastly, enhancing the text size in figures, particularly in Fig 3, is advised to improve legibility and overall clarity.
Response: The legibility and style of main Fig. 3 text sizes has been changed and additional figure formatting has been performed.
Referee 2
General: The manuscript by Losa et al., investigates whether ASC is involved in serum AA amyloidosis. The authors report that ASC colocalizes with SAA in human AA amyloidosis and that purified ASC specks accelerate SAA fibril formation in vitro. In addition, splenic AA amyloid was decreased in Pycard-/- mice compared to Pycard+/+ mice and that treatment with anti-ASC antibodies decreased amyloid loads in Pycard+/+ mice. Lastly, they analyzed serum of 19,334 patients to show that the prevalence of anti-ASC antibodies did not correlate with any specific disease. The authors conclude that ASC to play a role in extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases. The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies. However, there are number of issues that need to be addressed before acceptance for publication.
*Significance: The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies. This study reports a crucial role for ASC in SAA interaction and recruitment, SAA serum level modulation, SAA fibril formation acceleration, and controlling the extent of inflammation associated amyloidosis with respect to AA amyloid deposition *
Comment 1: Figure 3 E depicts Western blots of monomeric SAA in spleen of Pycard+/+ and Pycard-/- mice. The authors should include immunoblots depicting the levels of ASC in these tissues and to demonstrate that the Pycard-/- mice lack ASC.
Response: We did not perform ASC immunoblots for Pycard-/- and Pycard+/+ mice since the absence of the ASC protein in this well-established mouse line has been demonstrated in several key publications, including under inflammation conditions (right side of the figure below, from Mariathasan et al., Nature, 2014). However, we show ASC IHC of Pycard+/+ and Pycard-/- AA+ mice on spleen, confirming the absence of an ASC signal in Pycard-/- mice and its presence in the Pycard+/+ (Fig. 3F). Moreover, our genotyping data confirmed the presence and absence of the *Pycard *gene in Pycard+/+ and Pycard-/- AA+ mice.
Comment 2: Fig. 3B shows that at 96 hours after injection there was no difference in SAA serum concentration. How do the authors explain this drop in SAA serum concentration? No explanation is provided.
Response: Acute-phase response peaks at 24 hours after injury (i.e., Kushner I, 1982; Gabay et Kushner, 1999; Gitlin et Colten, 1987, Calif.: Academic Press, 1987:123-53)*. *Beyond 24 hours, acute phase proteins decay over time mirroring the process of tissue integrity restoration and the clearance of the insulting stimuli. This is in line with our data, where the inflammatory injury was induced by subcutaneous AgNO3 injection, resulting in a non-statistical serum SAA difference between the Pycard+/+ and Pycard-/- experimental mice at 96 hours post AgNO3 injection. In addition, the majority of SAA in Pycard+/+ mice was incorporated into amyloid deposit. As suggested by the reviewer we have included this explanation/references into the revised manuscript.
Comment 3: Figure 4 shows anti-ASC administration reduces amyloid load. The immunoblot in Figure 4C does not represent the quantification of the blot. In fact, there are only 3 samples per treatment group whereas the quantification shows 5-6 animals per group.
Response: We have performed two independent immunoblots at the same time to perform technical replicates (duplicates). As pointed out by the reviewer, this resulted in 6 samples and data points that were visualized and analyzed in main Fig. 4C. To avoid duplicating data, overloading the main figures with technical replicates, we opted to show only one representative immunoblot in the main Fig. 4C. The other blots are shown in the supplementary figures Fig. S13A and Fig. S13B for full transparency.
Comment 4: Additionally, the authors have not shown that the drug penetrates the target tissue and how much drug is present in spleen to provide a therapeutic effect. What is the half-life of the drug? These parameters are critical to assess the MOA of the anti-ASC used in these studies.
Response: To assess the pharmacokinetics of the anti-ASC antibody, we determined its titers in serum by ELISA at various time points up to 96 hpi after the first injection. The anti-ASC antibody serum levels peaked at 24 hpi and declined to about half maximal serum concentration levels at 96 hpi. This serum half-life, for the injected concentration, is in the range of reported kinetic parameters of engineered monoclonal antibodies (e.g., Unverdorben et al., MAbs, 2016; Foss et al., Nat Comm, 2024) (Fig. 4B). Because of the high permeability of splenic red pulp vasculatures, and because of the absence of any selectively permeable barrier, efficacious imbibement of the splenic extracellular space can be plausibly expected. Theoretically, one could perfuse mice intracardially with PBS and then measure antibody in tissue. Such measurements can work relatively well in the brain, which possesses a highly impermeable barrier. However, here we would find it difficult to convince ourselves that such measurements would not be contaminated by residual blood in splenic capillaries that may be difficult to clean up through perfusion. Therefore, we did not measure the antibody levels in the spleen.
Comment 5: The authors should expand the discussion section to include the work of other groups that have successfully employed anti-ASC antibodies. For example, PMID: 35793783, PMID: 32366256
Response: We thank the referee for pointing out that literature. We extended the discussion section accordingly and added these important references into the discussion.
Comment 6: Methods: The authors provide the number of animals employed in the Supplemental Tables 5 and 7. These numbers should be provided in the methods section or in the Figure legends. Additionally, how many replicates were performed for the data in Figure 2?
__Response: __As suggested by the reviewer we now provide the number of animals in the figure legends of main Fig. 2 and Fig. 3 in addition to those in Table 5 and Supp Table 7 to enhance clarity.
Referee 3____* *
General: The manuscript by Losa et al. explores the co-aggregation of ASC with serum amyloid A (SAA) in vivo and in mouse models, It posits that, similar to Amyloid beta, SAA is cross-seeded by ASC foci both in vitro and in vivo. This review only addresses the co-localization and in vitro cross seeding data (Figs. 1 and 2A, B), not the mouse experiments or mass spectrometry data. The manuscript first shows co-deposition of ASC with SAA amyloid. SAA was stained both with Congo red and ThS, both standard dyes for amyloid staining. Figure S2 shows CR birefringence, the hallmark of amyloid deposits. The authors then move to demonstrate co-localization of SAA and ASC in confocal and STED immuno-fluorescence microscopy.
Significance: The discovery of the role of ASC in Alzheimer's disease generated an exciting new hypothesis to the etiology of sporadic AD, for which the cause is unknown. The current manuscript finds that ASC may also play a role in AA amyloidosis, which is a significant finding.
Comment 1: Confocal images C-E show overlapping staining of markers for both SAA and ASC. Similarly, STED images show co-aggregation of ASC and SAA in amyloidosis patients. However, since confocal images F and G seem to show overlapping staining of the yellow and magenta channels as well, a careful quantitative analysis of the data I needed. Quantify co-localization (Pearson coefficient) in confocal and STED images. STED images from control patients are missing and need to be included.
Response: AA amyloidosis is a relatively rare disease, and tissue samples thereof are even rarer. We only had access to the samples of one patient in both control and SAA groups. This limitation prevented us from conducting quantitative analyses. Rather than looking at the Pearson – or, possibly better, Spearman – correlation coefficient, we opted for an unbiased method of correlation in which we reconstructed the picture using 3D surface rendering with the Imaris software (see Fig. 1). From this reconstruction, we exported the barycenter of each surface on a 3D plot for both SAA and ASC markers (see Fig. S2B-C). Each point represents the center of a surface, while the box plots on the sides represent the distribution of the markers in space, demonstrating the overlap of the markers for ASC and SAA. We also understand the suggestion to conduct STED imaging on control samples to show the absence of co-aggregation. However, we could not be sure of which region to capture and how to decide on the focus, as we did not detect strong signal from confocal images of the control sample. Imaging blindly would almost necessarily lead to irrelevant imaging and aberrant comparison. We do not claim any quantitative data out of these images; however, we report an observation. Quantitative and mechanistic co-aggregation data are presented in Fig. 2 using LiP-MS.
Comment 2: The authors then move on to demonstrate that ASC foci can cross-seed SAA amyloid formation in vitro, by recording SAA aggregation kinetics in the presence and absence of ASC foci. Curves recorded in the presence of ASC foci have accelerated kinetics as shown by a decrease in the time to reach half-maximal fluorescence (t1/2). However, these data (Fig 2A, B) are not very clean. Only three data points out of five curves shown in panel A. are presented in the fitting of the control (yellow) aggregation kinetics in panel B. Why was this done? Panel B shows a significant difference between the control and the kinetics seeded with ASC specks. It looks doubtful that the results are still statistically significant if these data are included, so their exclusion impacts the overall conclusion of the paper. The significance of the cross-seeding results needs to be substantiated experimentally.
__Response: __The in vitro SAA aggregation assay was performed under established conditions (Claus S et al., EMBO Rep 2017) and the resulting data was processed using the AmyloFit software from the Knowles lab in Cambridge, UK (Meisl G et al., Nat Protoc 2016). The AmyloFit technology uses global fitting resulting in high-accuracy kinetics. Given the software algorithm, only curves that show a sigmoidal ThT fluorescence signal over time can be fitted. Therefore, replicates that do not show aggregation (characteristic ThT signal) over time cannot be fitted. As a result, only three out of six curves could be fitted resulting in three t1/2. Conversely, in the presence of ASC specks, all six replicates aggregated in a dose-dependent manner, and could be fitted perfectly, yielding six t1/2 values. Thus, all available data points are plotted and used for statistical analysis. Moreover, the fact that in presence of ASC specks all SAA replicates aggregated/converted successfully in a dose-dependent manner (whereas in the SAA-only condition some replicates do not aggregate) further underscores the pivotal role of ASC specks in SAA seeding, conversion, and aggregation enhancement.
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Losa et al. explores the co-aggregation of ASC with serum amyloid A (SAA) in vivo and in mouse models, It posits that, similar to Amyloid beta, SAA is cross-seeded by ASC foci both in vitro and in vivo. This review only addresses the co-localization and in vitro cross seeding data (Figs. 1 and 2A, B), not the mouse experiments or mass spectrometry data.
The manuscript first shows co-deposition of ASC with SAA amyloid. SAA was stained both with Congo red and ThS, both standard dyes for amyloid staining. Figure S2 shows CR birefringence, the hallmark of amyloid deposits. The authors then move to demonstrate …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The manuscript by Losa et al. explores the co-aggregation of ASC with serum amyloid A (SAA) in vivo and in mouse models, It posits that, similar to Amyloid beta, SAA is cross-seeded by ASC foci both in vitro and in vivo. This review only addresses the co-localization and in vitro cross seeding data (Figs. 1 and 2A, B), not the mouse experiments or mass spectrometry data.
The manuscript first shows co-deposition of ASC with SAA amyloid. SAA was stained both with Congo red and ThS, both standard dyes for amyloid staining. Figure S2 shows CR birefringence, the hallmark of amyloid deposits. The authors then move to demonstrate co-localization of SAA and ASC in confocal and STED immuno-fluorescence microscopy.
Confocal images C-E show overlapping staining of markers for both SAA and ASC. Similarly, STED images show co-aggregation of ASC and SAA in amyloidosis patients. However, since confocal images F and G seem to show overlapping staining of the yellow and magenta channels as well, a careful quantitative analysis of the data I needed. Quantify co-localization (Pearson coefficient) in confocal and STED images. STED images from control patients are missing and need to be included. The authors then move on to demonstrate that ASC foci can cross-seed SAA amyloid formation in vitro, by recording SAA aggregation kinetics in the presence and absence of ASC foci. Curves recorded in the presence of ASC foci have accelerated kinetics as shown by a decrease in the time to reach half-maximal fluorescence (t1/2). However, these data (Fig 2A, B) are not very clean. Only three data points out of five curves shown in panel A. are presented in the fitting of the control (yellow) aggregation kinetics in panel B. Why was this done? Panel B shows a significant difference between the control and the kinetics seeded with ASC specks. It looks doubtful that the results are still statistically significant if these data are included, so their exclusion impacts the overall conclusion of the paper. The significance of the cross-seeding results needs to be substantiated experimentally.
Significance
The discovery of the role of ASC in Alzheimer's disease generated an exciting new hypothesis to the etiology of sporadic AD, for which the cause is unknown. The current manuscript finds that ASC may also play a role in AA amyloidosis, which is a significant finding.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
The manuscript by Losa et al., investigates whether ASC is involved in serum AA amyloidosis. The authors report that ASC colocalizes with SAA in human AA amyloidosis and that purified ASC specks accelerate SAA fibril formation in vitro. In addition, splenic AA amyloid was decreased in Pycard-/- mice compared to Pycard+/+ mice and that treatment with anti-ASC antibodies decreased amyloid loads in Pycard+/+ mice. Lastly, they analyzed serum of 19,334 patients to show that the prevalence of anti-ASC antibodies did not correlate with any specific disease. The authors conclude that ASC to play a role in extraneural proteinopathies of …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The manuscript by Losa et al., investigates whether ASC is involved in serum AA amyloidosis. The authors report that ASC colocalizes with SAA in human AA amyloidosis and that purified ASC specks accelerate SAA fibril formation in vitro. In addition, splenic AA amyloid was decreased in Pycard-/- mice compared to Pycard+/+ mice and that treatment with anti-ASC antibodies decreased amyloid loads in Pycard+/+ mice. Lastly, they analyzed serum of 19,334 patients to show that the prevalence of anti-ASC antibodies did not correlate with any specific disease. The authors conclude that ASC to play a role in extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases. The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies. However, there are number of issues that need to be addressed before acceptance for publication.
Major Points:
Figure 3 E depicts Western blots of monomeric SAA in spleen of Pycard+/+ and Pycard-/- mice. The authors should include immunoblots depicting the levels of ASC in these tissues and to demonstrate that the Pycard-/- mice lack ASC. Fig. 3B shows that at 96 hours after injection there was no difference in SAA serum concentration. How do the authors explain this drop in SAA serum concentration? No explanation is provided.
Figure 4 shows anti-ASC administration reduces amyloid load. The immunoblot in Figure 4C does not represent the quantification of the blot. In fact, there are only 3 samples per treatment group whereas the quantification shows 5-6 animals per group. Additionally, the authors have not shown that the drug penetrates the target tissue and how much drug is present in spleen to provide a therapeutic effect. What is the half-life of the drug? These parameters are critical to assess the MOA of the anti-ASC used in these studies.
The authors should expand the discussion section to include the work of other groups that have successfully employed anti-ASC antibodies. For example, PMID: 35793783, PMID: 32366256
Methods: The authors provide the number of animals employed in the Supplemental Tables 5 and 7. These numbers should be provided in the methods section or in the Figure legends. Additionally, how many replicates were performed for the data in Figure 2?
Significance
The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies. This study reports a crucial role for ASC in SAA interaction and recruitment, SAA serum level modulation, SAA fibril formation acceleration, and controlling the extent of inflammation associated amyloidosis with respect to AA amyloid deposition
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
In this manuscript, et al., investigates the role of the inflammasome adapter ASC (in AA amyloidosis). This condition involves the aggregation of serum amyloid A (SAA) and is linked to chronic inflammation.
Firstly, I can directly say that I do recommend this study for publication. This is a well conducted and well-written study which advances the knowledge on IL-1-independent inflammatory functions of inflammasomes. Furthermore, I find it particularly impressive that despite the inflammasome research community is well aware that amyloidosis is a hallmark of inflammatory diseases, it took a neuroscientist specialized in prion …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
In this manuscript, et al., investigates the role of the inflammasome adapter ASC (in AA amyloidosis). This condition involves the aggregation of serum amyloid A (SAA) and is linked to chronic inflammation.
Firstly, I can directly say that I do recommend this study for publication. This is a well conducted and well-written study which advances the knowledge on IL-1-independent inflammatory functions of inflammasomes. Furthermore, I find it particularly impressive that despite the inflammasome research community is well aware that amyloidosis is a hallmark of inflammatory diseases, it took a neuroscientist specialized in prion diseases to raise the question whether ASC would be involved in seeding serum AA aggregation.
Key findings include:
- ASC forms extracellular aggregates that enhance SAA aggregation, as observed through superresolution microscopy.
- In a mouse model, the absence of ASC significantly reduced amyloid load, not due to increased phagocytosis but likely due to diminished aggregation.
- Treatment with anti-ASC antibodies reduced amyloid load and mitigated weight loss in mice with AA amyloidosis.
These findings suggest that ASC plays a crucial role in AA amyloidosis and that targeting ASC could be a potential therapeutic strategy. The study expands our understanding of the involvement of ASC in proteinopathies beyond neural diseases, pointing to its role in systemic conditions like AA amyloidosis. Main Comments: Overall, the experiments are well-conducted and mostly all controls I would expect were included. With few exceptions, the data is convincing. With that said, I have issues with some of the staining employed in Fig 1.
In Fig. 1, the authors assess ASC staining in cardiac tissues from a patient with vasculitis and systemic inflammation-related AA amyloidosis, and a control patient who died of a heart attack but had no signs of amyloidosis. However, most of the data shown is related to the AL177 anti-ASC. More importantly, no isotype stainings are included. We have previously demonstrated that the AL177 anti-ASC, used here, reacts quite strongly with ASC−/− cells, and it is one of the less specific anti-ASC commercially available (PMID: 27221487). As this is data from one patient (understandably), I wonder if the authors could counterstain ASC in the same samples using a specific human anti-ASC with a different color (ex: Biolegend HASC), and confirm that the signal overlays with the AL-177.
Finally, in Figure 1H it seens from the description that another anti-ASC was used: "referred in the legend as ASC (MAB ASC, Yellow)". Is this a monoclonal anti-ASC? Also, the images show large and bright antibody aggregates (middle of the image, top left corner behind the "H", and a massive fluorescence in the bottom right of the image), indicating the presence of staining artifacts. Again, no counterstaining with isotype controls are shown.
Overall, although I don't dispute the possibility that ASC would co-localize with SAA deposits, I don't think the data presented can safely sustain that claim. I would, therefore, suggest that alternative methods to be employed to substantiate these conclusions: Supposedly, would it be possible to immuno-precipitate (IP) amyloid SAA and assess ASC via western blotting? As well as IP ASC and detect SAA? Or use DSS-crosslinking to find ASC oligomers in tissue areas rich in SAA?
Minor comments:
In addition to these main comments, some minor adjustments are recommended:
For example, it would be reasonable to quantify the results in Figure 3G and providing clarification regarding the controls in the figure legend. Though there is significantly less SAA in spleen homogenates from Asc−/−, there also seems to be the case for b-actin in Fig 3G. Moreover, in the figure legend the authors state: "...Spleen homogenate from untreated (-ctrl) and AA+ (+ctrl) C57BL/6 wt mice from an independent experiment served as negative and positive control, respectively." I don't know what the authors mean with that. Is this a montage, or samples from different experiments were run together in one blot? And if so, for what reason? This is confusing and should be clarified.
Furthermore, in the Abstract, a slight rephrasing is suggested to accurately describe ASC specks as molecular aggregates formed inside cells, which are subsequently released into the extracellular space.
Lastly, enhancing the text size in figures, particularly in Fig 3, is advised to improve legibility and overall clarity.
Significance
In conclusion, this manuscript offers valuable insights into the role of ASC in AA amyloidosis, presenting compelling findings that support its potential as a therapeutic target. Addressing the mentioned concerns and making the suggested revisions will further enhance the manuscript's scientific rigor and impact. Overall, this study is a valuable contribution to the field of inflammasome research and its relevance in systemic conditions like AA amyloidosis.
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