3 February 2024

The review on the manuscript, titled “Pharmacometabolomics approach identifies kynurenine as a
key marker of low dose interleukin-2 treatment in ALS” by Alacarn et al. submitted to the Annals of the New York Academy of Sciences.
Manuscript ID: annals—184

To Authors,

This paper titled “Pharmacometabolomics approach identifies kynurenine as a key marker of low dose interleukin-2 treatment in ALS” by Alacarn and colleagues investigates the metabolic changes induced by low-dose interleukin-2 (ld-IL-2) treatment in ALS patients and explores its correlation with regulatory T-cell (Treg) response. This study utilized samples from a randomized, placebo-controlled trial to evaluate ld-IL-2 levels in patients with ALS. Metabolomic profiling was performed using an AbsolutIDQ™ p180 kit, focusing on 188 metabolites. Statistical analyses, including univariate and multivariate methods, were used to assess metabolite variations and their relationship with Treg evolution. The results revealed distinct metabolic profiles between the treatment and placebo groups at different time points. Notably, an increase in kynurenine levels was observed in the treated group, consistent with ld-IL-2-induced activation of the kynurenine pathway. Baseline metabolome analysis predicted a Treg increase following ld-IL-2 treatment, suggesting potential metabolic markers of treatment response. Limitations, including the small sample size and the specificity of the targeted metabolomics approach, were acknowledged. Future directions include exploring the clinical response criteria and conducting larger-scale studies to validate the findings.
Overall, this study provides valuable insights into the metabolic alterations associated with ld-IL-2 treatment in ALS and highlights the potential of pharmacometabolomics in personalized medicine for neurodegenerative diseases.

In general, I think the idea of this article is really interesting, and the authors’ fascinating observations on this timely topic may be of interest to the readers of the Annals of the New York Academy of Sciences. However, some comments, as well as some crucial evidence that should be included to support the author’s argument, need to be addressed to improve the quality of the manuscript, its adequacy, and its readability prior to its publication in the present form. My overall opinion is to publish this research article after the author has carefully considered my comments and suggestions.

Please consider the following comments:
 In general, I recommend that the authors use more references to support their claims, especially in the Introduction section of the manuscript, which I believe is lacking. Thus, I recommend that the authors attempt to expand the topic of their article, as the bibliography is too concise; however, in my opinion, less than 30 research articles are too few. Therefore, I suggest that the authors focus their efforts on researching relevant literature. I believe that adding more studies and reviews will help provide a better and more accurate background for this study.
 Clarity and Structure: The paper is generally well written, but there are some instances where the text can be clearer and more concise. For example, in the introduction and results sections, detailed information is presented in a detailed manner. Breaking complex sentences and subheadings can enhance readability and comprehension.
 Introduction: It would be beneficial to include a brief discussion of the neural substrates underlying ALS pathology. ALS is characterized by progressive degeneration of the upper and lower motor neurons, leading to muscle weakness, atrophy, and eventual paralysis. While the exact etiology of ALS remains elusive, several key pathological mechanisms have been implicated, including aberrant protein aggregation, mitochondrial dysfunction, excitotoxicity, and neuroinflammation [1-2]. Understanding the complex interplay between these neural substrates is crucial for elucidating disease progression and developing effective therapeutic strategies [3-4]. Therefore, integrating insights from both systemic immune modulation and neural mechanisms provides a comprehensive framework for exploring the potential of ld-IL-2 as a therapeutic intervention for ALS.
 Justification of Methods: While the methods section is comprehensive, it would benefit from further justification of methodological choices. For example, why was the AbsolutIDQ™ p180 kit chosen for metabolomics analysis? Providing a rationale for method selection adds depth to the methodology and helps readers to understand why certain techniques are employed.
 Sample Size and Power: The study acknowledges the limited number of patients, which is an important consideration for the interpretation of results, especially in multivariate analyses. It would be helpful to discuss the potential impact of this limitation on the robustness of the findings and generalizability of the conclusions. In addition, considering the sample size, the statistical methods used should be appropriate and adequately powered.
 Interpretation of Results: The results section presents a comprehensive analysis of metabolomic profiles and their relationship with the Treg response. However, the interpretation of these findings could be strengthened by discussing the clinical relevance of the observed changes in metabolites. How do these metabolic alterations align with the known pathophysiological mechanisms of ALS and mode of action of ld-IL-2? Providing this context enhances the significance of results.
 Discussion of Limitations: While limitations are briefly mentioned in the discussion section, a more thorough discussion of study limitations and potential sources of bias would be beneficial. This could include factors such as the lack of external validation, specificity of the targeted metabolomics approach, and potential confounding variables.
 Future Directions: This paper briefly touches upon future perspectives, such as exploring clinical response criteria and conducting follow-up studies on larger cohorts. Expanding these future directions and discussing potential avenues for further research would provide valuable insights into the implications of the current findings and avenues for future investigation.
 Conclusion: The conclusions should succinctly summarize the key findings and their implications for personalized medicine in ALS treatment. It should also emphasize the novelty and significance of the study's contributions to the field.

Overall, this paper presents an interesting investigation into the metabolic changes associated with low-dose interleukin-2 (ld-IL-2) treatment in ALS patients and their correlation with regulatory T-cell (Treg) response.
I hope that after careful revision, the manuscript meets the journal’s high standards for publication. I declare no conflict of interest regarding this manuscript.

Best regards,

Reviewer

References:
1. https://doi.org/10.3390/ijms242115739
2. https://doi.org/10.3390/brainsci13081197
3. https://doi.org/10.1007/s00702-022-02513-5
4. https://doi.org/10.3390/biomedicines9101313
5. https://doi.org/10.3389/fnmol.2023.1217090