COVID-19 Induces Greater NLRP3 Inflammasome Activation in Obese Patients than Other Chronic Illnesses: A Case–Control Study

 Comments 1:

The article is interesting and addresses a relevant topic that has affected the world. I suggest some small improvements. Lines 52 to 54 contain a standalone sentence. Combine it with the paragraph to ensure better flow. Your introduction explaining how adipose tissue serves as a reservoir potentially increasing the viral load and facilitating the progression of the infection turned out well. In obesity, NLRP3 inflammasome activation is expected (as explained in line 59). In this study, no difference was observed between the groups, which is consistent given that both groups were obese. This highlights the absence of a non-obese group, which could potentially have shown lower NLRP3 activation compared to the obese groups.

Response 1:

Thank you for your thoughtful feedback and suggestions, which have been very important to refine our manuscript. In response to your comments, we have made several adjustments to address your observations.

As requested, we have adjusted the section between lines 52 and 54 to improve the flow of the text. The standalone sentence has been combined with the relevant paragraph, ensuring better coherence. Additionally, we restructured parts of the introduction, particularly to include the hypothesis regarding ASC and CASP-1, highlighting their roles in amplifying NLRP3 inflammasome activation. These changes can be found on page 02, between lines 52 and 74.

We also appreciate your positive feedback regarding the explanation of adipose tissue serving as a reservoir that potentially increases viral load and facilitates the progression of infection. This was a critical point in establishing the relationship between obesity and COVID-19 and framing the context of our study.

Regarding the absence of a non-obese control group, we acknowledge that this comparison could have been insightful. However, obesity is widely recognized as a strong inducer of NLRP3 inflammasome activation. Previous evidence has demonstrated that individuals with obesity exhibit chronic basal activation of the NLRP3 inflammasome, driven by persistent stimuli such as free fatty acids and oxidized lipoproteins, which sustain a low-grade inflammatory state (Bertocchi et al., 2020; López-Reyes et al., 2020; Unamuno et al., 2021; Vandanmagsar et al., 2011).

Considering that the activation of NLRP3 in obesity is well-documented in the literature, we opted to focus our study on comparing the magnitude of this activation in obese patients who died of severe COVID-19 to those with other critical, non-COVID-19 conditions. Our findings revealed that while both groups exhibited activation of the NLRP3 inflammasome, individuals with obesity and COVID-19 demonstrated significantly higher levels of ASC and CASP-1, suggesting a greater activation of this inflammatory pathway and a potential predominance of pyroptotic cell death (You et al., 2022; Frank & Vince, 2019).

We recognize the value of including a non-obese control group in studies exploring NLRP3 activation. However, given the extensive body of evidence already confirming the pro-inflammatory nature of obesity and its role in inducing NLRP3 inflammasome activation, we chose to focus on a clinically relevant question: how the activation of this pathway differs among obese individuals in the presence or absence of severe COVID-19. This approach allowed us to contribute new insights into the interplay between obesity and COVID-19 pathophysiology, without compromising the validity of our findings.

Comments 2:

As SARS-CoV-2 exacerbates NLRP3 activation, your hypothesis is that Group A would exhibit pronounced NLRP3 activation, as two factors would contribute to this: the condition of obesity itself and SARS-CoV-2. I believe you could include some hypotheses (including ASC e CASP-1) along these lines at the end of the introduction.

Response 2:

As requested, we have rewritten significant portions of the introduction to emphasize the role of ASC and CASP-1 in our study. Specifically, we included hypotheses regarding their involvement in amplifying NLRP3 inflammasome activation, particularly in the context of obesity and SARS-CoV-2 infection. These changes highlight the dual contribution of obesity-related chronic inflammation and the acute inflammatory insult caused by SARS-CoV-2, aligning with the hypothesis that Group A would exhibit pronounced NLRP3 activation due to these combined factors. The revised introduction incorporates these elements at its conclusion and can be found in page 02, between lines 52 and 74. We hope these adjustments meet the expectations and add clarity to the manuscript.

Comments 3:

Since your study is about obesity, I missed data on body mass index (BMI) and body fat to clearly show that the groups were indeed obese.

Response 3:

Thank you for your thoughtful observation regarding the inclusion of BMI and body fat data to ensure that the groups in our study were indeed classified as obese.

For Group A, all patients had numerical BMI data documented in their medical records, confirming their classification as obese. However, for Group B, the autopsy reports did not include exact numerical BMI values. Instead, these reports provided categorical classifications of nutritional status, such as cachexia, eutrophic, overweight, and obese. As detailed in the methodology, all autopsies in Group B were conducted prior to the pandemic, and the lung samples were obtained for various studies during that period.

Although we do not have the exact numerical BMI data for Group B, it is important to note that all patients included in the study had complete autopsy reports with adequate anthropometric classifications. Patients classified as cachectic, eutrophic, or overweight were excluded from the study, ensuring that only those classified as obese were included in Group B. This approach ensured consistency in the anthropometric criteria across both groups. To maintain clarity and transparency, we have included this distinction in the methodology and acknowledged it as a limitation of our study in page 9, line 266 to 272 and page 10, between lines 355 and 360.   

Comments 4:

Why is there no "CAUSE OF DEATH" column in Supplementary Table 1, as it is present in Table 2?

Response 4:

Thank you for raising this important point regarding the absence of a "Cause of Death" column in Supplementary Table 1. Group A, whose clinical and epidemiological characteristics are detailed in Supplementary Table 1, consists exclusively of individuals whose cause of death was severe COVID-19 or complications directly related to the infection. As all individuals in Group A shared the same cause of death, we determined that a separate "Cause of Death" column in Supplementary Table 1 was unnecessary. However, we value your observation and hope that this explanation aligns with the study's design and objectives.

Comments 5:

The panels of the Immunohistochemistry images could be better labeled, including numbers and letters, like in this example: "Immunohistochemistry images showing IL-6 staining in group A (1-left) and group B (2-right).

Response 5:

Thank you for your valuable suggestion. As requested, we have revised the figure captions and updated the labeling of the immunohistochemistry images to ensure greater clarity and consistency for the reader. These adjustments aim to make the presentation of results more intuitive and accessible for the audience.

The updated figure captions and image labels can be found on pages 03 and 4 (lines 105-110), 4 (lines 117-121) and 5 (lines 131-138). We believe these changes enhance the clarity of the results and better align with your recommendations.

Comments 6:

Your focus in the introduction was supported by the NLRP3 inflammasome pathway, but in the absence of differences, you tried to highlight your study in the discussion by emphasizing elevated levels of key protein structures such as ASC and CASP-1. I understand your effort, but it would be important to propose broader hypotheses in the introduction. In fact, ASC and CASP-1 (pyroptotic cell death) were not mentioned at all in the introduction. You need to better articulate what indicates greater NLRP3 activation with other proteins or pathways like ASC and CASP-1, as it seems you stretched the interpretation in the discussion, which in the introduction appeared to be simpler (a pure increase in NLRP3).

Response 6:

Thank you for your valuable feedback. We have carefully addressed your comments by revising the introduction to include more detailed information on the role of ASC and CASP-1 in the NLRP3 inflammasome activation pathway. Specifically, we expanded on the two key phases of inflammasome activation — priming and activation — to provide a more comprehensive explanation of how these proteins contribute to the pathway.

Additionally, as per your suggestion, we have included a hypothesis emphasizing the involvement of these key proteins, ASC and CASP-1, in the context of inflammasome activation, particularly under the dual influence of obesity-related chronic inflammation and the acute inflammatory stimulus caused by SARS-CoV-2. These changes aim to create a clearer alignment between the introduction and the discussion, addressing the potential mechanisms underlying the findings.

Furthermore, we have added a new and updated reference (2025) to the discussion section to further support our hypothesis that the differences observed between the two groups are likely due to the patients being in different phases of inflammasome activation. This additional reference provides recent insights into the regulation and dynamics of the NLRP3 pathway, reinforcing our interpretation.

These changes can be found on page 02, between lines 52 and 74 and on page 7, between lines 182 and 187. We hope these adjustments adequately address your recommendations and enhance the clarity, coherence, and scientific support of our manuscript.

Comments 7:

Some of the increased variables in Group B (IL-6, IL-18, CASP-9, and HIF) could be further explained.

Response 7: Thank you for your insightful comment regarding the increased levels of IL-6, IL-18, CASP-9, and HIF in Group B. We have carefully revised the discussion section of our manuscript and added further explanations to better contextualize these findings. Below, we summarize the key points that have been incorporated:

IL-6: We have expanded our discussion to emphasize that IL-6 is a well-established acute-phase cytokine. Given that the length of hospitalization was significantly shorter in Group B, this could partially explain the higher levels of IL-6 observed in this group. Since IL-6 tends to peak in the early stages of inflammation and then decrease over time, the prolonged hospitalization of COVID-19 patients in Group A might have contributed to lower IL-6 levels at the time of sample collection. Added in: Page 8, Lines 224-229.

IL-18: We have highlighted that, in addition to being a cytokine directly linked to obesity, IL-18 has activation pathways beyond the NLRP3 inflammasome. These alternative pathways, such as protease-mediated cleavage, could explain its elevated levels in Group B, independent of inflammasome activation. This reinforces the idea that IL-18 expression is influenced by multiple regulatory mechanisms beyond SARS-CoV-2 infection. Added in: Page 9, Lines 254-260.

CASP-9: We have provided additional details regarding the role of caspase-9 in cell death pathways. The elevated concentration of caspase-9 in Group B, combined with the reduced levels of caspase-1 in this group, further supports our hypothesis that the NLRP3 inflammasome pathway is more active in Group A. This suggests that the predominant cell death mechanism in the control group is likely mediated by caspase-9 rather than caspase-1, reinforcing the idea that inflammasome activation plays a more significant role in COVID-19 cases. Added in: Page 7, Lines 158-164.

HIF: We have expanded our discussion on the baseline conditions of patients in Group B and provided additional evidence supporting the activation of the HIF-1α pathway in these individuals. Several comorbidities present in Group B, such as hepatic cirrhosis, thromboembolic events, and metastatic neoplasms, are known to induce HIF-1α activation. This could explain why HIF levels were markedly elevated in the control group, even when compared to the COVID-19 group, which also had multiple factors contributing to increased HIF expression. Added in: Pages 9 and 10, Lines 300-310.

We hope these additions provide a more comprehensive understanding of the observed differences between groups.