NLRP3 Inflammasome and IL-1–Mediated Inflammation in Human Carotid Atherosclerosis: A Systematic Review of Endarterectomy-Based Evidence
, Antónia Rocha-Melo-Sousa 1,2,*,†, Mohammed Shahat 3, Cármen Tavares 4, Lina Carvalho 5, Vitor Sá-Martins 4, Manuel Neiva-Sousa 6,7, Mariana Fragão-Marques 8 and João Rocha Neves 9,10,11Round 1
Reviewer 1 Report
Comments and Suggestions for Authorssee attached file
Comments for author File: 
Comments.pdf
Author Response
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Comments 1:
This manuscript addresses an important and timely topic: the role of the NLRP3 inflammasome and IL-1-mediated inflammation in carotid plaque vulnerability and outcomes after carotid endarterectomy. The topic is biologically relevant, clinically interesting and suitable for publication. The authors also attempt to integrate plaque biology, circulating biomarkers and postoperative outcomes, which is a strength. However, in its current form, the article needs major revision before publication: R: We sincerely thank the reviewer for the thorough and constructive evaluation of our manuscript, and for recognizing the biological relevance and clinical interest of the topic
1. The title and objective suggest a review of the impact of NLRP3/IL-3 mediated inflammation on outcomes after carotid endarterectomy, yet many included studies do not actually examine postoperative outcomes after CEA, several are mainly plaque biology studies, non-outcome biomarker studies or not strictly CEA-only populations. The manuscript includes ex vivo-in vitro studies. Thus, this creates a mismatch between the title, the aim, the eligibility criteria. Thus, my suggestion is to retitle and restructure the manuscript as, for example, “NLRP3 inflammasome and IL-1 mediated inflammation in human carotid atherosclersosis: evidence from endarterectomy based studies”. However, if the authors want to preserve the “outcomes after carotid endarterectomy” emphasis, then they should include only studies that truly report: - Perioperative stroke/death/MI - - - - Restenosis Recurrent neurologic events Long-term MACE Survival after CEA. R: We agree that the original title and stated objective created a genuine mismatch with the scope of the included evidence. Upon reflection, we have decided to adopt the reviewer's suggested retitling, which we consider more accurate and intellectually honest given the nature of the studies included: "NLRP3 Inflammasome and IL-1–Mediated Inflammation in Human Carotid Atherosclerosis: A Systematic Review of Endarterectomy-Based Evidence”
Several included studies date back to 2000, 2008, 2009, 2012, and 2013. The review would benefit from the inclusion of more recent studies, as the current evidence base should better reflect contemporary advances in carotid imaging, biomarker profiling, molecular pathology, and perioperative management. A stronger emphasis on updated literature would improve the clinical relevance and translational value of the manuscript. In this regard, the authors may consider including more contemporary references, such as the study published in Diagnostics (DOI: 10.3390/diagnostics13081478), which may provide relevant updated insights into carotid plaque assessment and biomarker-based risk stratification.
R: We thank the reviewer for this observation and appreciate the suggestion of additional contemporary literature. We wish to clarify that our systematic search was conducted in January 2026 and captured all eligible publications available up to that date across three databases (PubMed/MEDLINE, Scopus, and Web of Science). Any newly identified eligible studies have been assessed against the pre-specified inclusion criteria and incorporated into the revised manuscript where appropriate. Regarding the older studies included in this review, we wish to note that their inclusion reflects the results of a comprehensive and unbiased systematic search rather than a selective literature approach. These studies represent the earliest human investigations of the NLRP3/IL-1 axis in carotid endarterectomy specimens and provide a foundational mechanistic and translational context that more recent work builds upon. Excluding them on the basis of publication date alone would introduce a form of recency bias inconsistent with systematic review methodology. We have, however, taken the reviewer's broader point on board and have strengthened the Discussion to more explicitly contextualize older findings within the framework of contemporary advances in carotid imaging, biomarker profiling, molecular pathology, and perioperative management. We have inserted the reviewer's specific reference (DOI:10.3390/diagnostics13081478).
2. Tables The tables are useful but need editorial refinement. - - Table 2: A large amount of missing data limits usefulness. Consider simplifying or moving to supplement if not central. R: We acknowledge that the substantial proportion of missing data in Table 2 limits its interpretive value in the main text. We include Table 2 in the main text for two reasons.
The pattern of missing data itself is informative. It demonstrates systematic reporting gaps across the included studies and directly substantiates our key findings regarding methodological heterogeneity and evidence comparability. This transparent presentation of the data's limitations contributes meaningfully to the manuscript.
Second, the demographic and comorbidity context in Table 2 is essential for readers to assess generalizability in section 3.3. The table's placement in the main text serves the interpretive needs of the results discussion. Supplementary placement would disperse this critical context and require additional qualification in the text. Table 3: This is probably the most valuable table, but it should distinguish: • tissue vs blood biomarkers, • direct NLRP3/IL-1 markers vs indirect associated markers, • semiquantitative vs quantitative assays.
R: We thank the reviewer for this observation and fully agree that the proposed distinctions would substantially improve the analytical value of Table 3, which we likewise consider the most informative table in the manuscript. We have revised Table 3 to incorporate the three suggested distinctions as follows:
A new column — "Assay Type" — has been added, with entries categorized as:
This distinction is particularly important for interpreting the comparability of findings across studies and for contextualizing the limitations of cross-study synthesis, and has been explicitly referenced in the revised narrative of section 3.3.
Table 3 now explicitly identifies the biological compartment for each biomarker measurement, with entries consistently categorized as Carotid plaque tissue, Plasma, Serum, Peripheral blood mononuclear cells (PBMCs), or combinations thereof.
- Tables 4 and 5: These show how sparse the outcome data are, but they may be better reframed as: • studies reporting perioperative outcomes • studies reporting follow-up events rather than appearing as complete datasets The manuscript includes Tables 4 and 5 on perioperative and long-term outcomes, but most entries are “not reported” or “NA.” My suggestion is to explicitly state: “Most included studies were not designed to evaluate clinical outcomes after CEA; therefore, evidence regarding postoperative events remains sparse and indirect.” This would improve the honesty and clarity of the review. R: We fully agree that the current presentation of Tables 4 and 5 creates a misleading impression of completeness, when in reality the sparse outcome data reflect a fundamental characteristic of the included evidence base rather than a reporting gap in our extraction process.
The following statement has been added to the limitations section: "Most included studies were not designed to evaluate clinical outcomes after CEA. The majority were primarily plaque biology, biomarker, or mechanistic investigations in which CEA served as the tissue acquisition platform rather than the index intervention under study. Therefore, evidence regarding perioperative and long-term postoperative events remains sparse, and the findings presented in these tables should be interpreted accordingly."
3. One sentence is awkward: “Moreover, information for adequate pathology routine evaluation of endarterectomy specimens might guide patient follow-up.” This should be rewritten more clearly. R: The sentence was revised. Now you can read:
“Furthermore, standardised pathological evaluation of endarterectomy specimens, informed by the evidence synthesised here, may provide actionable insights to guide postoperative patient follow-up and risk stratification.” |
Reviewer 2 Report
Comments and Suggestions for AuthorsOverall, the manuscript is well-structured, clearly written and addresses an important translational gap between molecular mechanisms and clinical outcomes. However, several methodological, reporting, and interpretative issues limit the strength and clarity of the conclusions.
First, the manuscript tends to overinterpret associations, particularly regarding prognostic value of IL-1/NLPR3 biomarkers, and the impact of PCSK9 inhibitors. Also, the distinction between association vs prediction vs causation is not always clearly maintained. Another example is when the authors cited the Marfella et al, whose study is observational, yet the language in article implies causal therapeutic benefit.
Another issue is that the manuscript should clearly justify its inclusion criteria in a clinical systematic review both of ex vivo/in vitro studies, or maybe separate them into a dedicated subsection, if possible.
The narrative synthesis occasionally reads as a list of findings rather than a synthesis, especially in Results section 3.3.
Minor issues: table 3 is very detailed but difficult to read; also, several tables contain excessive "NA" values, which reduces interpretability; there are also inconsistent formatting.
Recommendation for improvement:
- Provide full search strategy in main or appendix
- Clarify inclusion/exclusion decisions
- Group findings more clearly (e.g., tissue vs systemic vs outcomes)
- Reduce descriptive listing
- Avoid causal language
- Emphasize uncertainty and limitations more strongly
While overall language is good, there are multiple issues: repetition (“Xie et al. Xie et al.” (duplicate)); typographical errors; awkward phrasing: overly long sentences in Discussion reduce readability.
Author Response
Overall, the manuscript is well-structured, clearly written and addresses an important translational gap between molecular mechanisms and clinical outcomes. However, several methodological, reporting, and interpretative issues limit the strength and clarity of the conclusions.
R:
We thank the reviewer for acknowledging the manuscript's structure and relevance to translational research.
First, the manuscript tends to overinterpret associations, particularly regarding prognostic value of IL-1/NLPR3 biomarkers, and the impact of PCSK9 inhibitors. Also, the distinction between association vs prediction vs causation is not always clearly maintained. Another example is when the authors cited the Marfella et al, whose study is observational, yet the language in article implies causal therapeutic benefit.
R: We agree that the original manuscript did not always maintain a sufficiently rigorous distinction between association, prediction, and causation, and we have revised the text accordingly throughout. We have replaced overstated language (e.g., "predicts", "determines") with appropriately hedged terminology (e.g., "is associated with", "may reflect", "warrants further investigation as a potential prognostic marker").
The revised manuscript now explicitly frames these findings as hypothesis-generating rather than practice-defining, and we have added the following qualifier: "Whether pharmacological modulation of the NLRP3/IL-1 axis through PCSK9 inhibition translates into clinically meaningful benefit in the CEA population remains to be established in prospective, interventional studies."
Marfella et al. is an observational cohort study, and the original manuscript used language that implied a causal therapeutic relationship.
We have revised this passage to read: "suggesting a potential mechanistic link, though causality cannot be inferred from this study design."
Another issue is that the manuscript should clearly justify its inclusion criteria in a clinical systematic review both of ex vivo/in vitro studies, or maybe separate them into a dedicated subsection, if possible.
R: The authors agree and propose a new structure
3.3.1 — NLRP3/IL-1 in atherosclerotic plaques (clinical/observational studies only) 3.3.2 — Mechanistic insights from ex vivo and in vitro investigations (Monaco, Potor, Wang) 3.3.3 — Systemic biomarkers (current 3.3.2) 3.3.4 — Short-term outcomes 3.3.5 — Long-term outcomes 3.3.6 — Upstream and downstream relationships
As these studies do not contribute patient-level clinical data, their findings are presented separately and interpreted as a mechanistic context (Monaco, Potor, Wang).
The narrative synthesis occasionally reads as a list of findings rather than a synthesis, especially in Results section 3.3.
R: We thank the reviewer for this observation, which reflects a genuine limitation of the original draft. We agree that several subsections of section 3.3 presented findings sequentially by author rather than synthesising them around interpretive themes, patterns of convergence, or outstanding gaps.
“Taken together, the systemic biomarker data converge on a consistent pattern: circulating and intracellular markers of NLRP3/IL-1 activation track plaque vulnerability across different biological compartments (peripheral blood mononuclear cells and plasma) and across methodologically distinct approaches (12)(22) . Notably, this systemic activation appears to persist beyond the surgical event itself, as Profumo et al. demonstrated elevated monocytic IL-1β up to six months post-CEA, suggesting that CEA removes the local trigger but not the underlying systemic inflammatory state (17) . Whether this residual activation represents a therapeutic target or merely a marker of systemic atherosclerotic burden remains unanswered by the available evidence.”
Minor issues: table 3 is very detailed but difficult to read; also, several tables contain excessive "NA" values, which reduces interpretability; there are also inconsistent formatting.
R: We thank the reviewer for these observations regarding table presentation and formatting.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for addressing the requested revisions and for the improvements made to the manuscript. In my opinion, the article may now be accepted for publication in its current form.
