Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Almost all NLRP3 reviews have focused on its inhibition with respect to neuro-degeneration, whereas this review also considers the complex role of NLRP3 in cancer, being either helpful or harmful. The clear simple summary to encourage readers new to the field is important.

Section 7d: Thymoquine and OLT1177 are not the most selective or potent NLRP3 inhibitors?   

Are there other NLRP3 inhibitors with more up to date relevant references? The references below maybe useful to consider newer inhibitors of NLRP3 (with better BBB penetration), although studies are largely focused on neuroinflammation?:

https://doi.org/10.1021/acs. jmedchem.3c02098.

 https:// doi.org/10.1021/acs.jmedchem.4c02350. 

 

Minor corrections:

line 101: Delete 'he'

line 153: I think 'section' should be 'secretion'.

line 252: These (not Those)

line 277: of, not if.

line 318-320: rewrite sentence....increase in activation of NLRP3 causes higher levels of IL-1b.

Fig 2: NE7 should be NEK7

Ref 28: delete '2024'

 

Author Response

 

Reviewer 1

Comment: Almost all NLRP3 reviews have focused on its inhibition with respect to neuro-degeneration, whereas this review also considers the complex role of NLRP3 in cancer, being either helpful or harmful. The clear simple summary to encourage readers new to the field is important.

Section 7d: Thymoquine and OLT1177 are not the most selective or potent NLRP3 inhibitors?  

Are there other NLRP3 inhibitors with more up to date relevant references? The references below maybe useful to consider newer inhibitors of NLRP3 (with better BBB penetration), although studies are largely focused on neuroinflammation?:

https://doi.org/10.1021/acs. jmedchem.3c02098.

https:// doi.org/10.1021/acs.jmedchem.4c02350.

Response: We thank the reviewer for this insightful comment. We agree that while OLT1177 is a significant clinical candidate, newer small molecule inhibitors offer superior potency and, crucially, better Blood-Brain Barrier (BBB) penetration as an essential factor given this review’s focus on the neurodegeneration-cancer axis.

As suggested, we have revised the Section 7d skin cancer section to include more potent inhibitors such as MCC950 (the gold standard for potency) and newer, BBB-permeable compounds like Inzomelid and NP3-109, citing the suggested recent literature (PMID: 38175811; PMID: 39574318). We have also added a nuance regarding the pharmacokinetic requirements for treating neuroinflammation versus systemic malignancies.

Minor corrections:

line 101: Delete 'he'

Response: Corrected as suggested

line 153: I think 'section' should be 'secretion'.

Response: Corrected as suggested

line 252: These (not Those)

Response: Corrected as suggested

line 277: of, not if.

Response: Corrected as suggested

line 318-320: rewrite sentence....increase in activation of NLRP3 causes higher levels of IL-1b.

Response: Sentence rewritten

Fig 2: NE Corrected as suggested7 should be NEK7

Response: Changed from NE7 to NEK7

Ref 28: delete '2024'

Response: Date '2024' deleted

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a broad and ambitious narrative review of the role of the NLRP3 inflammasome in both neurodegeneration and cancer, highlighting its context-dependent and often paradoxical functions. The topic is relevant and well aligned with current interest in innate immunity, chronic inflammation, and cross-disease mechanisms. The authors have compiled an extensive body of literature and cover a wide range of molecular pathways, disease models, and therapeutic strategies.

However, the manuscript is very long, highly descriptive, and at times lacks sufficient synthesis and critical evaluation. Several sections read more like a textbook-style overview than a focused review article. In addition, there are issues related to language clarity, repetition, imprecise statements, and inconsistent depth across disease sections. Substantial revision is needed to improve structure, focus, and readability, and to better distinguish well-established evidence from speculative or model-dependent findings.

Major Comments

Q1. What is the central conceptual message of this review beyond the general statement that NLRP3 acts as a “double-edged sword,” and could this be stated more clearly in the Introduction and reinforced throughout the manuscript?

Q2. Given the breadth of topics covered, how do the authors justify the current length and level of detail, and could some sections (e.g., historical background and basic inflammasome biology) be shortened to improve focus?

Q3. Many disease-specific sections are largely descriptive; could the authors strengthen the manuscript by more explicitly contrasting conflicting findings and discussing sources of inconsistency across studies (e.g., model systems, cell types, disease stage)?

Q4. In the cancer sections, how do the authors distinguish between tumor-intrinsic versus immune-cell–derived NLRP3 signaling, and should this distinction be more clearly emphasized to avoid oversimplification?

Q5. The review frequently implies therapeutic potential of NLRP3 modulation; could the authors more clearly separate preclinical evidence from clinical data and avoid overinterpreting animal or in vitro findings?

Q6. In the neurodegeneration sections, how do the authors position NLRP3 activation relative to other key pathogenic processes (e.g., protein aggregation, mitochondrial dysfunction) in terms of causality rather than association?

Minor Comments

Q7. The manuscript contains repeated explanations of inflammasome structure and activation; could these be consolidated to reduce redundancy?

Q8. Several statements are written in a very general or informal manner; would careful language editing improve precision and scientific tone throughout the manuscript?

Q9. Figures 1 and 2 are helpful conceptually; could adding key references to the figure legends improve their standalone value?

Q10. Table 1 is extensive but difficult to navigate; would adding clearer references or subdividing the table improve readability?

Q11. Some disease sections (e.g., certain cancers) rely on a small number of studies; could the authors clarify where conclusions are based on limited evidence?

Q12. Would a brief summary table comparing neurodegeneration versus cancer (cell types involved, dominant cytokines, therapeutic implications) help synthesize the core message?

Author Response

Reviewer 2

Q1. What is the central conceptual message of this review beyond the general statement that NLRP3 acts as a “double-edged sword,” and could this be stated more clearly in the Introduction and reinforced throughout the manuscript?

Response: We appreciate the reviewer’s request for a more defined conceptual framework. While "double-edged sword" describes the outcome, the central conceptual message we aim to convey is the context-dependent threshold hypothesis. We argue that the pathological vs. protective nature of NLRP3 is determined by three specific variables- (1) the cellular source (myeloid vs. tumor cell), (2) the cytokine ratio (IL-1β vs. IL-18 predominance) and (3) the mode of cell death (homeostatic clearance vs. inflammatory pyroptosis).

We have revised the Introduction and other sections in the manuscript to explicitly state this framework and have updated the section headings and concluding remarks of each chapter to reinforce how these specific variables dictate the "polarity" of the NLRP3 response in both neurodegeneration and oncology. (Page No.- 2-3, 8 and 20, highlighted in Red)

 

Q2. Given the breadth of topics covered, how do the authors justify the current length and level of detail, and could some sections (e.g., historical background and basic inflammasome biology) be shortened to improve focus?

Response: We appreciate the reviewer's suggestion to improve the manuscript's focus. However, we believe that a focused summary of NLRP3 biology remains scientifically essential for this specific review. To understand the ‘double-edged sword’ effect, one must grasp the nuanced differences between Signal 1 (priming) and Signal 2 (activation), as many pro-tumorigenic effects are linked to chronic priming, while anti-tumorigenic effects (pyroptosis) are linked to acute activation. Furthermore, these biological details are necessary to justify the therapeutic checkpoints section, as each drug mentioned targets a specific molecular stage discussed in the biology section. We have carefully reviewed the text and implemented a significant reduction in the "Historical Background" and "Basic Inflammasome Biology" sections. We believe the revised, more concise version successfully balances foundational clarity with thematic focus.

 

Q3. Many disease-specific sections are largely descriptive; could the authors strengthen the manuscript by more explicitly contrasting conflicting findings and discussing sources of inconsistency across studies (e.g., model systems, cell types, disease stage)?

Response: We agree that the double-edged nature of NLRP3 often arises from experimental and biological variables that can lead to seemingly conflicting results. In the revised manuscript, we have transitioned from a descriptive summary to a more analytical comparison. We now explicitly discuss sources of inconsistency, addressing how NLRP3 activation in early-stage disease differs from chronic late-stage activation (pathological) and cell-type polarity contrasting the effects of NLRP3 in myeloid cells versus tumor-intrinsic signaling. We have added some insights in both the cancer and neurodegeneration sections to provide a more nuanced interpretation of the current literature. (Page No.- 8 and 10, highlighted in Red)

 

Q4. In the cancer sections, how do the authors distinguish between tumor-intrinsic versus immune-cell–derived NLRP3 signaling, and should this distinction be more clearly emphasized to avoid oversimplification?

Response: The spatial compartmentalization of NLRP3 signaling specifically whether it originates from the tumor cell (intrinsic) or the infiltrating myeloid cells (extrinsic) is a primary determinant of its eventual ‘polarity’. We have revised the oncology sections to explicitly categorize signaling into tumor-intrinsic and immune-derived pathways. Specifically, we now emphasize that tumor-intrinsic NLRP3 often promotes EMT and chemoresistance, whereas immune-derived NLRP3 can drive either potent anti-tumor Th1 responses (via IL-18) or pro-tumorigenic immunosuppression (via IL-1β), depending on the tumor microenvironment (Sauter et al., 2024; Derangère et al., 2014). (Page no.- 7, highlighted in Red)

 

Q5. The review frequently implies therapeutic potential of NLRP3 modulation; could the authors more clearly separate preclinical evidence from clinical data and avoid overinterpreting animal or in vitro findings?

Response: While we did include dedicated sections for ‘Targeting NLRP3’ and ‘Clinical Trials’, we agree that the language used to describe in vitro and in vivo findings should more explicitly reflect their preclinical nature to avoid overinterpretation. We have revised the ‘Therapeutic Potential’ section to clearly delineate between findings in immortalized cell lines (e.g., PC3, U138MG), murine models, and human clinical data. Furthermore, we have tempered our conclusions by using terms such as ‘preclinical proof-of-concept’ and ‘putative therapeutic target’, when discussing non-human data. We have also added a clarifying paragraph at the beginning of that section to emphasize that the transition from bench to bedside remains challenging due to the lack of physiological complexity in standard 2D cell cultures. (Page No.- 13, highlighted in Red)

 

Q6. In the neurodegeneration sections, how do the authors position NLRP3 activation relative to other key pathogenic processes (e.g., protein aggregation, mitochondrial dysfunction) in terms of causality rather than association?

Response: To demonstrate causality rather than mere association, we now emphasize three key pieces of evidence on the pre-onset activation- post-mortem and imaging data showing NLRP3 activation in mild cognitive impairment (MCI) and early-stage PD, indicating it precedes gross neuronal loss; and interventional rescue- evidence that NLRP3-deficient mice are significantly protected against the seeding of Tau and α-synuclein, proving that the inflammasome is required for the prion-like spread of these proteins (3) The feed-forward loop- we describe how NLRP3-derived ASC specks act as physical scaffolds that cross-seed Aβ and α-synuclein, establishing a causal vicious cycle, where inflammation directly accelerates protein aggregation. However, we have revised the neurodegeneration section to move beyond the associative presence of NLRP3 and instead emphasize its role as a fundamental driver of disease progression. (Page No.- 10-11, highlighted in Red)

 

Minor Comments

Q7. The manuscript contains repeated explanations of inflammasome structure and activation; could these be consolidated to reduce redundancy?

Response: We have conducted a thorough revision of those sections to eliminate redundant definitions of the protein domains and general innate immune signaling. However, we have opted to maintain the distinct separation between structural architecture and the two-step activation process, as this framework is scientifically fundamental to our double-edged sword thesis. We believe that retaining specific mechanistic details is required for a rigorous understanding of the mode of action of NLRP3 in the following contexts such as differentiating disease polarity, mechanistic linkage to neurodegeneration and justifying therapeutic checkpoints.  We have streamlined the prose to ensure it is focused and non-repetitive while preserving the specialized terminology and molecular steps that serve as the connective tissue between the bench-side mechanism and the clinical phenotype.

 

Q8. Several statements are written in a very general or informal manner; would careful language editing improve precision and scientific tone throughout the manuscript?

Response: We have thoroughly revised the manuscript to improve language precision and ensured a more consistent scientific tone. Statements that were previously expressed in an informal manner were rewritten with more precise wording and terminology.

 

Q9. Figures 1 and 2 are helpful conceptually; could adding key references to the figure legends improve their standalone value?

Response: We have updated the legends for Figure 1 (Polarity of NLRP3) and Figure 2 (Activation cascade and checkpoints) to include primary citations that support the bimodal signaling outcomes and the specific molecular checkpoints. Specifically, we have added references covering the dichotomy of IL-1β/IL-18 (e.g., Ghiringhelli et al., 2009) and the structural activation steps involving NEK7 and GSDMD (e.g., He et al., 2016; Shi et al., 2016). This allows the figures to serve as standalone summaries for the reader.

 

Q10. Table 1 is extensive but difficult to navigate; would adding clearer references or subdividing the table improve readability?

Response: We agree table 1 was extensive and difficult to navigate. To improve readability, we reorganized the table into clearly defined sections based on disease category (neurodegenerative, cancer, and inflammatory diseases), streamlining the wording to mechanistic descriptors that are ensured by references. These changes should improve the clarity of the table.

 

Q11. Some disease sections (e.g., certain cancers) rely on a small number of studies; could the authors clarify where conclusions are based on limited evidence?

Response: We revised the relevant disease specific sections to explicitly indicate where conclusions are based on a limited number of available studies. In these cases, we clarified the strength of the evidence and avoided using overgeneralized wording. We also highlighted these conclusions in a clear and transparent manner and emphasized the need for cautious interpretation where data is limited.

 

Q12. Would a brief summary table comparing neurodegeneration versus cancer (cell types involved, dominant cytokines, therapeutic implications) help synthesize the core message?

Response: We would like to highlight that Table 1 (The role of the NLRP3 inflammasome in various cancer and neurodegenerative diseases) was specifically designed and updated for this purpose. This table provides a high-level scientific synthesis by contrasting the disease-specific triggers (e.g., Aβ/Tau in neurodegeneration vs. ATP/P2X7R in cancer), the shared downstream enzymatic effects (Caspase-1/GSDMD), and the diverging pathogenic outcomes (e.g., neuronal death in PD/AD vs. proliferation and immunosuppression in Lung/Breast cancer). By categorizing these elements side-by-side, Table 1 allows the reader to immediately grasp the bimodal nature of NLRP3, where chronic neuroinflammation drives cell loss, while similar inflammatory outputs in the tumor microenvironment facilitate niche expansion and metastasis. We believe this existing table effectively serves the synthesis role requested without requiring a redundant second table.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their answers and revision.

Author Response

Thank you for reviewing our revised manuscript and for the positive feedback.

Also, we have addressed the specified corrections- the term "Snail" has been capitalized in all instances related to snail signaling and highlighted in Red. "ASC oligonization" has been corrected to "ASC oligomerization" in Figure 2.