Fluoxetine Reshapes Macrophage Membrane Sphingolipids and Inflammatory Response Without Affecting Extracellular Vesicle Biogenesis upon Inactivated SARS-CoV-2 Stimulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors focus on investigating the regulatory effects of FTX on sphingolipid metabolism, EV biogenesis, and inflammatory responses in THP-1 cells, aiming to explore its potential role in alleviating virus-induced hyperinflammation. Through lipidomic, EV, and inflammatory cytokine analysis, the authors attempt to identify the ASM–Cer–S1P axis as a key target through which FTX modulates sphingolipid metabolism. However, the current sphingolipidomic data suffers from incomplete coverage and insufficient presentation. Although the authors observed that FTX alters the size of EVs derived from THP-1 cells and speculated that such alterations may be associated with functional changes, this hypothesis remains unverified.

Despite touching upon an intriguing intersection between FTX, sphingolipid metabolism, and inflammation regulation, the study exhibits several notable limitations that undermine the credibility and scientific impact of its conclusions. Nonetheless, this study provides preliminary insights into the interaction between FTX and sphingolipid metabolism in the context of inflammation, laying a potential foundation for future investigations into novel anti-inflammatory strategies. While the findings are exploratory, they merit further development to enhance their robustness and translational relevance. Therefore, I recommend that the authors address the following points by supplementing key experimental data, clarifying ambiguities, and correcting formatting issues.

 

1. The authors report that FTX significantly increases S1P levels, yet S1P is generally considered a pro-inflammatory mediator. The manuscript does not discuss how this observation aligns with the proposed anti-inflammatory mechanism of FTX.
2. There is a lack of direct evidence demonstrating altered expression or activation of SMases. Notably, neutral sphingomyelinase (nSMase), rather than acid sphingomyelinase (aSMase), is primarily responsible for SM hydrolysis at the plasma membrane. Given that the study focuses on aSMase while omitting necessary controls or discussion regarding nSMase, the interpretation of results appears incomplete.
3. In Figure 2, C23:1 and C25 are not among the major canonical SM species. Moreover, the authors do not specify whether these are d18-sphinganine, d18:1-sphingosine, or d16 variants. When using polar head groups as the basis for MRM transitions, distinguishing among sphingomyelin species can be analytically challenging. Additionally, the significant change in C23:1 levels in both untreated and SARS-CoV-2-exposed macrophages is unexpected for a non-canonical SM species; the authors should provide a rationale or interpretation for this finding.
4. In Figure 1, portions of the text are too small to read clearly, compromising figure readability.
5. Some specialized terms (e.g., “FIASMA,” “lipid rafts”) lack concise definitions upon their first appearance, creating potential comprehension barriers for readers unfamiliar with the field.
6. Transitions between subsections in the Discussion are abrupt (e.g., the sudden shift from “sphingolipid metabolism” to “EV biogenesis”), with no logical connectors or bridging statements, which disrupts the flow of the narrative.
7. Minor formatting and typographical errors are present throughout the manuscript (e.g., unnecessary line breaks in the authors' affiliations, absence of superscript notation in concentration expressions such as “10⁻⁶ M”), which detract from overall readability (e.g., Figures 1B and 1C).
8. The study is limited to in vitro experiments using THP-1 cells, with no in vivo validation (e.g., in SARS-CoV-2-infected animal models), thereby limiting the translational applicability of the conclusions.
9. FTX was only tested at a single concentration of 1 µM, with merely a comparison between 10⁻⁷ M and 10⁻⁶ M. A full dose–response curve is absent, preventing assessment of dose-dependency or identification of an optimal concentration for modulating sphingolipid metabolism and inflammation.
10. Key methodological details are missing. For instance, detailed information about critical reagents (e.g., source, catalog number, and batch number) has not been provided—including for FXT and ELISA kits (BD OptEIA™, R&D DuoSet)—which hinders reproducibility.
11. Parameters for acceleration and deceleration during ultracentrifugation (using the Beckman Coulter 70 Ti rotor) were not specified, despite the fact that these settings can critically influence EV yield and purity.
12. The linkage between subpanels E/F/G/H of Figure 4 and the LPS-stimulated condition is not clearly explained in the figure legend, leading to potential confusion.
13. Statistical annotations in Figure 4 are ambiguous: p-values are missing in several instances, and the specific group comparisons represented by certain p-values are not clarified in the legend.
14. The schematic in Figure 4I oversimplifies the ASM–Cer–S1P axis and fails to explain the mechanistic basis for the observed increase in S1P levels, potentially leading to misinterpretation of FTX's anti-inflammatory action.
15. The translational implications of the findings are overstated. The manuscript claims that FTX “holds therapeutic potential in viral infection-associated excessive inflammation,” yet this assertion is not sufficiently substantiated. No in vivo or clinical data are presented, and the conclusion rests solely on in vitro observations in THP-1 cells.
16. The hypothesis that reduced EV size correlates with functional changes remains unverified. The authors speculate that altered EV size “may change their functions,” but no proteomic or lipidomic profiling of EV cargo or functional assays using recipient cells were performed. This renders the claim speculative and unsupported by empirical evidence.

Author Response

Response to Reviewer 1 Comments:

Comments and Suggestions for Authors

Despite touching upon an intriguing intersection between FTX, sphingolipid metabolism, and inflammation regulation, the study exhibits several notable limitations that undermine the credibility and scientific impact of its conclusions. Nonetheless, this study provides preliminary insights into the interaction between FTX and sphingolipid metabolism in the context of inflammation, laying a potential foundation for future investigations into novel anti-inflammatory strategies. While the findings are exploratory, they merit further development to enhance their robustness and translational relevance. Therefore, I recommend that the authors address the following points by supplementing key experimental data, clarifying ambiguities, and correcting formatting issues.

 We sincerely appreciate your positive feedback and thoughtful evaluation of our manuscript. We are pleased that you found our study well-conducted and insightful. Your comments are highly valuable for improving our work, and we look forward to addressing any specific suggestions you may have.

1. The authors report that FTX significantly increases S1P levels, yet S1P is generally considered a pro-inflammatory mediator. The manuscript does not discuss how this observation aligns with the proposed anti-inflammatory mechanism of FTX.

Response: Indeed, sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator whose biological effects are highly context-dependent and can be either pro- or anti-inflammatory depending on the cellular compartment, receptor engagement, and inflammatory milieu. While extracellular S1P signaling has been associated with pro-inflammatory responses in certain settings, particularly in lymphocyte trafficking and vascular inflammation, this interpretation cannot be generalized across all cell types; studies indicate that in macrophages S1P accumulation, especially in the context of reduced ceramide levels, can be associated with pro-survival, immunoregulatory, and inflammation-resolving functions [1–4].

In macrophages, S1P has been increasingly recognized as a regulator of cell survival, metabolic adaptation, and immune homeostasis [5,6]. Importantly, intracellular pools of S1P exert signaling functions that differ substantially from those of extracellular S1P, which acts in a cytokine-like manner through autocrine or paracrine receptor activation [7]. In the present study, S1P levels were quantified and no evidence of enhanced inflammatory signaling was observed downstream. On the other hand, the increase in S1P induced by FTX was accompanied by a consistent reduction in classical pro-inflammatory mediators, suggesting that S1P accumulation in this context reflects metabolic reprogramming rather than inflammatory activation. Moreover, the functional outcome of S1P signaling is influenced by the balance between ceramides and S1P, commonly referred to as the ceramide/S1P rheostat [8,9]. Although receptor-specific signaling through S1P receptors was not directly assessed in this study, the overall sphingolipidomic and functional profile supports a model in which S1P elevation occurs as part of a coordinated immunometabolic adaptation that dampens macrophage inflammatory responses rather than exacerbating them [10-12].

 

[1] Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol. 2003;4:397–407. https://doi.org/10.1038/nrm1103

[2] Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu Rev Immunol. 2012;30:69–94. https://doi.org/10.1146/annurev-immunol-020711-075011

[3] Obinata H, Hla T. Sphingosine 1-phosphate and inflammation. Int Immunol. 2019 Aug 23;31(9):617-625. doi: 10.1093/intimm/dxz037

[4] Joshi JC, Joshi B, Rochford I, Mehta D. S1P Generation by Sphingosine Kinase-2 in Recruited Macrophages Resolves Lung Inflammation by Blocking STING Signaling in Alveolar Macrophages. J Cell Signal. 2021;2(1):47-51.

[5] Chiappa NF, Lal N, Botchwey EA. Resolving versus non-resolving sphingolipid dynamics during macrophage activation: a time-resolved metabolic analysis. J Lipid Res. 2025 Nov;66(11):100899. doi: 10.1016/j.jlr.2025.100899

[6] Lee M, Lee SY, Bae YS. Functional roles of sphingolipids in immunity and their implication in disease. Exp Mol Med. 2023 Jun;55(6):1110-1130. doi: 10.1038/s12276-023-01018-9

[7] Hait NC, Allegood J, Maceyka M, et al. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science. 2009;325:1254–1257. https://doi.org/10.1126/science.1176709

[8] Quinville BM, Jin Z, et al. A Comprehensive Review: Sphingolipid Metabolism and Rheostat. International Journal of Molecular Sciences. 2021;22(11):5793. https://doi.org/10.3390/ijms22115793

[9] Maceyka M, Spiegel S. Sphingolipid metabolites in inflammatory disease. Nature. 2014; 510:58–67. https://doi.org/10.1038/nature13475

[10] Jernigan PL, Makley AT, Hoehn RS, Edwards MJ, Pritts TA. The role of sphingolipids in endothelial barrier function. Biol Chem. 2015 Jun;396(6-7):681-91. doi: 10.1515/hsz-2014-0305

[11] Abboushi N, El-Hed A, El-Assaad W, et al. Ceramide inhibits IL-2 production by preventing PKC-dependent NF-κB activation. J Immunol. 2004;173:3193–3200. https://doi.org/10.4049/jimmunol.173.5.3193

[12] Gulbins E, Palmada M, Reichel M, et al. Acid sphingomyelinase–ceramide system mediates effects of antidepressant drugs. Nat Med. 2013;19:934–938. https://doi.org/10.1038/nm.3214

 

 

2. There is a lack of direct evidence demonstrating altered expression or activation of SMases. Notably, neutral sphingomyelinase (nSMase), rather than acid sphingomyelinase (aSMase), is primarily responsible for SM hydrolysis at the plasma membrane. Given that the study focuses on aSMase while omitting necessary controls or discussion regarding nSMase, the interpretation of results appears incomplete.

Response: The focus of the present study on aSMase was intentional and guided by the pharmacological mechanism of action of FIASMAs, which selectively induce functional inhibition of aSMase through lysosomal destabilization rather than directly targeting neutral sphingomyelinase (nSMase). Although nSMase is classically associated with sphingomyelin hydrolysis at the plasma membrane, substantial evidence demonstrates that SARS-CoV-2 infection critically depends on aSMase-driven ceramide platform formation. In particular, Carpinteiro et al. showed that SARS-CoV-2 induces rapid activation of aSMase, leading to ceramide-enriched membrane domains that promote ACE2 clustering and viral entry, and that pharmacological inhibition of aSMase (example, amitriptyline, ambroxol) markedly reduces infection [1,2].

To address the potential contribution of nSMase-dependent pathways, we included GW4869, a selective nSMase inhibitor, as a functional control in EV assays. While GW4869 reduced EV production as expected, FTX did not alter EV release, indicating that the effects of FIASMAs observed in our model are independent of nSMase-mediated exosome biogenesis. Together, our data support that the biological effects are primarily associated with aSMase inhibition and sphingolipid remodeling rather than nSMase-dependent mechanisms. We have clarified this rationale and the complementary role of nSMase inhibition in the revised Discussion section.

 

[1] Carpinteiro A, Gripp B, Hoffmann M, Pöhlmann S, Hoertel N, Edwards MJ, et al. Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells. J Biol Chem [Internet]. 2021;296:100701. Available from: https://doi.org/10.1016/j.jbc.2021.100701.

[2] Carpinteiro A, Edwards MJ, Hoffmann M, Kochs G, Gripp B, Weigang S, et al. Pharmacological Inhibition of Acid Sphingomyelinase Prevents Uptake of SARS-CoV-2 by Epithelial Cells. Cell Reports Med [Internet]. 2020;1(8). Available from: https://doi.org/10.1016/j.xcrm.2020.100142.

 

3. In Figure 2, C23:1 and C25 are not among the major canonical SM species. Moreover, the authors do not specify whether these are d18-sphinganine, d18:1-sphingosine, or d16 variants. When using polar head groups as the basis for MRM transitions, distinguishing among sphingomyelin species can be analytically challenging. Additionally, the significant change in C23:1 levels in both untreated and SARS-CoV-2-exposed macrophages is unexpected for a non-canonical SM species; the authors should provide a rationale or interpretation for this finding.

Response: We thank the reviewer for this insightful comment. Odd-chain sphingolipids, as well as other odd-chain lipid species, are indeed present at relatively low concentrations in humans, as extensively reported in plasma and tissues [1]. These fatty acids are primarily derived from dietary sources, particularly ruminant milk and meat, and are subsequently incorporated into human lipid metabolism [2]. Further, in cell culture these lipids could be incorporated by the use of Serum Bovine Fetal  (SBF) to the cell medium. Although their basal levels are generally lower than those of even-chain sphingolipids, this does not preclude their biological relevance. Importantly, accumulating evidence indicates that odd-chain sphingolipids are associated with metabolic and inflammatory disorders and may serve as potential biomarkers in several pathological conditions [3–5].

Regarding the analytical strategy, our MRM method was not based exclusively on polar head group fragments. For each sphingolipid species, we employed specific precursor-to-product ion transitions, incorporating both class-specific and species-specific fragment ions. In addition to monitoring the diagnostic phosphocholine fragment (m/z 184.07 for SM), we confirmed lipid identity using characteristic fragmentations, neutral losses, and chromatographic retention times. This multi-parameter approach increases analytical confidence and minimizes ambiguity in species assignment. A detailed description of this methodology has been previously published by our group [6]. To further support our assignments, we now provide representative raw MS/MS spectra for SM d18:1/23:1 and SM d18:1/25:1, illustrating precursor ions, diagnostic head-group fragments, and characteristic neutral losses:

 

 

We acknowledge that the sphingoid base composition was not sufficiently detailed in the original version. All sphingomyelin species detected in our targeted lipidomic analysis were based on a d18:1 sphingosine backbone, with exceptions corresponding to sphinganine species. This information has now been explicitly incorporated into the revised text and figures.

        

[1] Quehenberger, O. et al. Lipidomics reveals a remarkable diversity of lipids in human plasma1. J. Lipid Res. 51, 3299–3305 (2010).

[2] de Carvalho, L. P. et al. Plasma Ceramides as Prognostic Biomarkers and Their Arterial and Myocardial Tissue Correlates in Acute Myocardial Infarction. JACC Basic Transl. Sci. 3, 163–175 (2018).

[3] Toro, D. M. et al. Plasma Sphingomyelin Disturbances: Unveiling Its Dual Role as a Crucial Immunopathological Factor and a Severity Prognostic Biomarker in COVID-19. Cells 12, 1938 (2023).

[4] Mundra, P. A. et al. Large-scale plasma lipidomic profiling identifies lipids that predict cardiovascular events in secondary prevention. JCI Insight 3, (2018).

[5] Ye, J. et al. Targeted lipidomics reveals associations between serum sphingolipids and insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. Diabetes Res. Clin. Pract. 173, (2021).

[6] Felippe, T. V.D. et al. High-resolution targeted mass spectrometry for comprehensive quantification of sphingolipids: clinical applications and characterization of extracellular vesicles. Anal. Biochem. 698, 115732 (2025).

 

4. In Figure 1, portions of the text are too small to read clearly, compromising figure readability.

Response: We agree with the reviewer's concern and we modified the main figure.

 

5. Some specialized terms (e.g., “FIASMA,” “lipid rafts”) lack concise definitions upon their first appearance, creating potential comprehension barriers for readers unfamiliar with the field.

Response: In the revised Introduction section, we included concise definitions of specialized terms at their first occurrence to improve accessibility for non-specialist readers. Specifically, we clarified: “Lipid rafts are specialized membrane microdomains enriched with cholesterol and lipids, which characterizes to be are very heterogeneous and highly dynamic, as well as to be responsible to compartmentalize several cellular process (https://www.jlr.org/article/S0022-2275(20)33205-3/fulltext ; https://journals.biologists.com/jcs/article/136/9/jcs260887/308924/The-role-of-lipid-rafts-in-vesicle-formation ; https://pubmed.ncbi.nlm.nih.gov/32093594/ )”.

In addition, we have now provided a concise definition of FIASMA (functional inhibitors of acid sphingomyelinase) at their first mention, highlighting their relevance in modulating sphingolipid metabolism and membrane organization.

 

6. Transitions between subsections in the Discussion are abrupt (e.g., the sudden shift from “sphingolipid metabolism” to “EV biogenesis”), with no logical connectors or bridging statements, which disrupts the flow of the narrative.

Response: We have carefully revised this section and incorporated explicit bridging statements and conceptual connectors to better link sphingolipid metabolism, membrane organization, and EV biogenesis. These additions provide a clearer conceptual continuity between subsections, improving the overall coherence and readability of the Discussion.

 

7. Minor formatting and typographical errors are present throughout the manuscript (e.g., unnecessary line breaks in the authors' affiliations, absence of superscript notation in concentration expressions such as “10⁻⁶ M”), which detract from overall readability (e.g., Figures 1B and 1C).

Response: We agree with the reviewer’s observation and appreciate this careful assessment of the manuscript.

 

8. The study is limited to in vitro experiments using THP-1 cells, with no in vivo validation (e.g., in SARS-CoV-2-infected animal models), thereby limiting the translational applicability of the conclusions.

Response: We fully agree that in vivo validation would further strengthen the translational relevance of our findings. The present study was designed as a mechanistic and hypothesis-generating investigation, focusing on well-controlled in vitro experiments using THP-1-derived macrophages, a widely established model for studying inflammatory and lipid metabolic responses in the context of viral infections. This experimental approach allowed us to systematically dissect the effects of SARS-CoV-2 stimulation and FIASMA treatment on sphingolipid remodeling and EVs biogenesis under defined conditions, minimizing confounding variables inherent to in vivo models.

Nevertheless, we acknowledge that in vivo studies are essential to validate and extend these observations in a physiological context, and will be issued in the next project. Accordingly, we have expanded the Discussion section to explicitly address this limitation and to emphasize the need for validation in relevant animal models and clinical settings.

 

9. FTX was only tested at a single concentration of 1 µM, with merely a comparison between 10⁻⁷ M and 10⁻⁶ M. A full dose–response curve is absent, preventing assessment of dose-dependency or identification of an optimal concentration for modulating sphingolipid metabolism and inflammation.

Response:  Although FTX was used at 1 µM in the main experiments, its concentration was selected based on an initial dose-screening analysis, now included in the Supplementary Information, and on concentrations commonly employed in previously published studies. In this assay, cells were treated with FTX across a range of concentrations (10⁻⁷ to 10⁻⁵ M), consistent with doses reported in the literature for in vitro studies. The highest concentration tested (10⁻⁵ M) significantly reduced cell viability and was therefore excluded from subsequent analyses. Importantly, intermediate concentrations (10⁻⁷ and 10⁻⁶ M) produced comparable biological effects, with no statistically significant differences between them. Based on these results and prior reports, we selected 10⁻⁶ M (1 µM) for subsequent experiments, as it represents the highest non-cytotoxic concentration that consistently modulated sphingolipid metabolism.

 

10. Key methodological details are missing. For instance, detailed information about critical reagents (e.g., source, catalog number, and batch number) has not been provided—including for FXT and ELISA kits (BD OptEIA™, R&D DuoSet)—which hinders reproducibility.

Response: We appreciate your suggestions and have incorporated the changes into the Materials and Methods section text, as well in the supplementary material.

 

11. Parameters for acceleration and deceleration during ultracentrifugation (using the Beckman Coulter 70 Ti rotor) were not specified, despite the fact that these settings can critically influence EV yield and purity.

Response:  We have revised this point accordingly in the methods section.

 

12. The linkage between subpanels E/F/G/H of Figure 4 and the LPS-stimulated condition is not clearly explained in the figure legend, leading to potential confusion.

Response: We have incorporated the necessary corrections into the figure legend text.

 

13. Statistical annotations in Figure 4 are ambiguous: p-values are missing in several instances, and the specific group comparisons represented by certain p-values are not clarified in the legend.

Response: The legend now explicitly describes the experimental groups being compared in each panel, and brackets clearly indicate the corresponding pairwise comparisons between stimulated conditions (SARS-CoV-2 or LPS) in the absence or presence of fluoxetine (FXT). In addition, we have ensured that all reported statistically significant differences are accompanied by their corresponding p-values. As stated in the revised legend, only statistically significant comparisons (p < 0.05) are displayed, while non-significant comparisons were omitted for clarity and visual readability.

 

14. The schematic in Figure 4I oversimplifies the ASM–Cer–S1P axis and fails to explain the mechanistic basis for the observed increase in S1P levels, potentially leading to misinterpretation of FTX's anti-inflammatory action.

Response: To address this concern, we have revised Figure 4I. The updated schematic now reflects the dynamic balance between ceramide generation, sphingosine formation, and S1P production. Furthermore, we have expanded the corresponding section of the Discussion to better contextualize these metabolic pathways and to clarify how aSMase inhibition by FXT may indirectly promote S1P accumulation through modulation of downstream enzymatic activities and substrate availability. These revisions provide a more comprehensive and mechanistically accurate framework for interpreting the effects of FXT on sphingolipid signaling and inflammatory responses.

 

15. The translational implications of the findings are overstated. The manuscript claims that FTX “holds therapeutic potential in viral infection-associated excessive inflammation,” yet this assertion is not sufficiently substantiated. No in vivo or clinical data are presented, and the conclusion rests solely on in vitro observations in THP-1 cells.

Response:  We agree that the translational implications were overstated. Accordingly, our findings should be interpreted as mechanistic and hypothesis-generating rather than as direct evidence of therapeutic applicability. In response to the reviewer’s concern, we have carefully revised the manuscript to adopt more cautious and balanced language. Specifically, statements referring to “therapeutic potential” have been replaced with wording that emphasizes FTX as a modulator of inflammatory and sphingolipid pathways in vitro, and that highlights the need for further validation in relevant in vivo and clinical models.

 

16. The hypothesis that reduced EV size correlates with functional changes remains unverified. The authors speculate that altered EV size “may change their functions,” but no proteomic or lipidomic profiling of EV cargo or functional assays using recipient cells were performed. This renders the claim speculative and unsupported by empirical evidence.

Response: In response to this concern, we have revised the relevant sections of the manuscript to clearly frame these observations as preliminary and hypothesis-generating. Statements suggesting that altered EV size “may change their functions” have been reworded to emphasize their speculative nature and to explicitly acknowledge the absence of direct functional validation. Furthermore, we have expanded the Discussion to highlight the need for future studies integrating EV cargo characterization and recipient-cell functional assays to validate these hypotheses.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article under the title:

 “Fluoxetine reshapes macrophage membrane sphingolipids and infmammatory responses without affecting extracellular vesicle biogenesis upon inactivated-SARS-CoV-2 stimulation” by J. C.S de Carvalho and co-authors aimed at exploring whether FXT can re-shape sphingolipidome of macrophages by inhibiting ASM-Cer axis. They also measure levels of two Ils and two MMPs, and size distribution of Evs from maxcrophages. While a sound sound experimental work lies behind the study, it has several serious issues that must be addressed.

The authors may want to play down the hype on modulation of sphingolipidome, when they presented only eleven ceramides, ten SMs, 4 sphingoid bases and S1P. In the big lipidomic era when we whitness big hundreds of lipids and few dozens of SL species, deciphered at the level of known base + known FA residue, the study before me cannot draw significant conclusions on alterations in SL metabolism based on their results.

I would certainly not support the expression: ”we provide mechanistic evidence that FXT-induced remodeling of the Cer pathway” without the proper explanation of ceramide pathway (whatever it may be).

”The observed increase in SM and S1P levels, accompanied by reduced Cer species, is consistent with pharmacological inhibition of ASM activity.” It may be, but it also may be due to an increased activity of sphingomyelin synthase, enzyme that converts Cer to SM.

The authors omitted to explain how they link increase in S1P with decrease in Cer and increase in SM. They write about Cer as it is a single lipid entity, when it is known that, while one Cer species may increase, the other may decrease. They should perform pathway enrichment analysis and try to link the matching species, suhc as how decreased levels of C18 Cer matches increased levels of C18 SM, and do the same for C20 Cer and C20 SM, C24 Cer and C24 SM, etc. This way, functional enrichment analysis would recognize patterns in altered enzyme activities, since, for example, ASM cannot change sphingoid base or FA residue in SM when converting it into Cer, it can only cleave phosphocholine from SM. In other words, ASM cannot produce C14 Cer from C16 or C18 SM.

The Discussion is oversimplified, reflecting the lack of lipidomic expertise. For example: ”SM participates in multiple cellular processes, including cell division...”. The authors talk about SM as if it is a single lipid species with uniform role, while there are hundreds of SM species and some could have opposing roles, e.g pro-inflammay+tory and anti-inflammatory.

How can you be sure that only acid sphingomyelinase is involved in Cer drop and SM rise? Are there any other Smases at the membrane? Would they be able to interact with FXT? Can you try an assay which would demonsrate the binding:

Also want to see PCA plots.

The lack of expertise in lipid species, their nature, nomenclature and enzymatic conversions is obvious.

I want to see  clear comments on alterations in Cer and SM; levels in Figures 2A, 2B and 2C, comparing stimulated and non-stimulated groups, FXT-treated vs non-treated etc. The authors gave only two vague sentences, when combined with poor grammar and generalized conclusion (which belong to the Discussion) I learnt nothing. It is not readers job to analyse the Figures and draw conclusions.

-authors tend to use generalized conclusions (hypotheses) not really based on data obtained, such as: ”Indeed, the notable S1P increase following FXT treatment, these findings support an anti-apoptotic and anti-inflammatory environment.”

Much, more experiments should be done in order to conclude this.

Also, ”The TEM analyses revealed that treatment with FXT substantially reduces the average diameter of EVs released after stimulation with SARS-CoV-2 particles, indicating that FXT treatment modulates cargo composition and diminishes the EVs size.” EV size and size distribution was analysed on TEM, but, cargo composition was not.

-line 307: One cannot conclude that ”attenuating the production of inflammatory mediators induced by different pathogenic stimuli” simply based on one stat. significant change in IL-6 and one pathogenic stimulus (LPS).

-The manuscript leved an impression that it was hastily written, without a critical deep reading. I noticed that authors commented on Figures 4 in 3.3, but the plots and pictures were in Figure 3. This is not a sole example. In the subsection 3.4. the authors discuss on Figure 4, again.

-The experiment with LPS treatment of macrophages was not even mentioned in Methods.

I like the picture 4I.

 

 

Other issues:

-line 55: not only in macrophages are lipid rafts enriched in cholesterol...

-line 61: not all SLs are bioactive  lipids, actually most of them are structural components of cell membranes, especially sphingomyelins. Biaoctive lipids are sphingoid bases (S1P), ceramides, ceramide-1-phosphate (Cer1P) and several others.

-line 65: De novo SLs biosynthesis...

-line 68-70: Why are authors talking about ceramide in singular? It should be ceramides (as well as sphingomyelins), since many dozens of different fatty acyl residues (FA) can be attached to sphinganines (giving rise to dhCer) or sphingosines (giving rise to ceramides), not to mention sphingoid bases with different number of C-atoms such as C16-sphingosine, C18 sphingosine, C19 sphingosine, C20 sphingosine, etc.

-line 69: HexCer is glycosyl ceramide, since Hex can be Glc or Gal. If you are sure that it was glucose, the name is glucosyl ceramide (GlcCer), and if you are sure that it was galactose, the name is galactosyl ceramide (GalCer). When not sure about the nature of the hexose, then, the name is hexosyl ceramide (HexCer).

-line 70: LacCer is lactosyl-ceramide (Hex2Cer), since Lac consist of one Glc and one Gal.

-line 71: many lipid species can be considered as key signaling lipids, so let us change this into: S1P, a key lipid involved in immune cell functions

-line 77: which Cer species promotes the listed functions? It is written in singular, so I cannot tell which one? Let em give you an examples, C16-Cer has a pro-inflammatory effect promoting insulin resistance, activvates NF-kappaB, thus controbuting to metabolic syndrome, whereas C24-Cer suppresses inflammatory signaling and protects against tissue damage, correlating with reduced risk of cardiovascular disesase. In the light of these facts, one cannot simply put dozens of different CERAMIDES into one lipid with one function.

-line 80: please inform us first what is LPS; then use the abbreviation

-line 81: I wish the authors could be a bit more specific when writing about: ”with discrete metabolites correlating with distinct activation states [8].”

 Which are these discrete metabolites?ž--line 87 is a bit wordy, I could not understand what is promoter of what? Do ceramides, produced from SMs by SMases, drive or promote driving? What do you mean by: ”driving of intraluminal vesicles withing MVB?”

-Line 89: please use different term from the ”lipid vesicles”, since they are membraneous structures but also carry non-lipid cargo or?

-line 93: what is the difference between EXTRACELLULAR and intercellular microenviroment? They are both outside the cell. Also, the expression: ”supporting the physiological processes” is so wide, as literally everything occurring inside the body is covered by this expression. Please be more specific when writing about the roles of Evs. Which processes?

-line 96: please writeh what is ESCRT pathway

Line 100: you can remove it, as you have already mention changes in SLs composition in line 97, and since roles in neurodegeneration, cardiovascular disease, and cancer progression are not in your line of research presented in this manuscript. The introduction is too long. You may want to stick to inflammation caused by viral diseases

-line 104: please delete : ”an enzyme responsible for Cer production”, it is a repetition (line 71). Also it may sound ambiguous in this context, since there are other enzymes that produce Cer.

-Line 106, please explain the abbreviation FIASMA

-line 107: please check the grammar...”such as diminishes”, should be: ...”such as diminishing Cer-rich membrane domains”

-line 110: chronic FXT treatment of what? Which cells?

-line 113: should be: ”...and, potentially, susceptibility of cells to viral infection”

-line 114-116: be aware of the grammar, when you mention FXT along with SARS-CoV-2, then it is vague which of the two is under: ”ITS broader immunomodulatory…”

-line 117: be aware of the grammar: ...inactivated particles induces...(plural)

-line 118: it would be nice to know which are these ”key pro-inflammatory mediators commonly elevated in COVID-19”

-line 119, please be more specific, tell us which cellular membranes are mentioned in : ”remodels the SLs composition in cellular membranes”

-line 120: be aware of the grammar, it is vague what you meant in: ”affected under these conditions”. Which are these conditions? COVID infection or FTX treatment, since both of the two were mentioned in the previous sentence.

-line 122: please change into: ” …we provide new evidence of FTX repurposing in regulating”. Delete the word: ”collectively”, as I don’t understand what is here collectively, all of the authors together doing the study or?

-line 123: please explain: ” …membrane lipid metabolism”. Is there specific membrane lipid metabolism apart from the common lipid metabolism, or did you mean ”membrane lipid re-organization”

-line 124: please change into: ” …beyond being an anti-depressant, towards”

-line 129:…for the prophylactic exposure…

-line 130:…following THE treatment…to remove THE residual drug

-line 132: it is very difficult to undersand the study design without the sentence telling us that control differentiated macropohages were not treated with FXT, but later were stimulated by SARS CoV2. I wish you could just simply tell us that you had 4 groups: i) THP-1 macrophages treated with FXT and stimulated by virus (MφV-FXT), ii) THP-1 macrophages not treated with FXT, but stimulated by virus (MφV), iii) THP-1 macrophages not treated with FXT and not stimulated by virus (double-negative control, Mφ0) and iv) THP-1 macrophages treated with FXT but not stimulated by virus (Mφ0-FXT).

-line 136:…for THE subsequent EVs isolation…

Please use MACROPHAGES insetad of CELLS, wherever possible, to avoid confusion

-line 139: please delete the: ”For SLs extraction” and start with: 1×10⁶ macrophage cells were suspended…since the subtitle says it all

We would need the full noun of PBS here

I need to know why did you add HCL in the extraction solvent (Folch, chloroform/Methanol 2/1)?

-line 143:…and THE second extraction…please avoid wordiness and use shorter sentences, like this one: ”after the organic phase was collected, we re-extracted the aqueous phase with another 2 mL of CHCl3.”

We perfomed extraction = we extracted

We perfomed analysis = we analysed etc etc…

-line 144 should read: ”combined organic phase with the extracted lipids was evaporated…then resuspended in…”(when you combine the two, you get one combined solution)

-line 155: ”Lipids were analysed using a TripleTOF 5600+ mass spectrometer”

I corrected this since you did not only DETECT lipid species (qualitative analysis), you quantified them (quantitative analysis=measuring lipid concentrations/abundances)

-line 180: all capital letters Extracellular Vesicles Characterization

-line 184:…for THE morphological assessment…

-line 188:…visualized using a JEM-2100…

-lines 134 and 189, duplication of FMRP-USP. What is it?

-line 190: please change the title into Quantification of protein mediators

-line 191: be aware of the grammar: ...secretion of...were quantified (should be singular). Also you measured concentration of cytokines and MMPs and not secretion. Hence, write: We measured cytokines: IL-10....as well as MMPs:

-line 199: please be careful of the correct terms. You should write: ”Multivariate analyses were conducted using…”. Lipidomic analysis is when you perform qualitative and quantitative analysis of the lipid species in your samples, and this can be done only by LC MS/MS. Then you use the columns with pmol/mL values for these lipids and input it into software tools such as MetaboAnalystR, ggplot2 and ComplexHeatmap. That is called DATA analysis, and since you have many variables (measure lipids) it is also called multivariate data analysis. Therefore, you cannot perform Multivariate lipidomics analyses using R packages. R packages see data solely as numbers (data is data, regardless their origin).

-line 201: what do you mean under ”parametric comparisons”? I know lipid species are usually non-normally distributed, did you check for these distributions prior to ANOVA?

line 207: please shorten into: ” Figure 1A depicts how FXT treatment affected the sphingolipidome in THP-1-derived macrophages. ” It is all sadi in this short sentence.

I corrected this because when you write: ”…modulate macrophage lipid profiles”, one expects to see the total lipidome of macrophages, that is phospholipids, glycerolipids etc, not only SLs.

-line 210, please use SL instead of sphingolipid

-line 210: I dont understand the meaning of: ” similar overall sphingolipid profiles, despite the intensity of Cer and SM on MφV group”. What is exactly the intensity of Cer and SM in group? All measured ceramides or one specific Cer species (same for SM)?

Why didnt you acquaint us about the different doses of FTX applied to the studied groups in Methods? It seems all of a suddent to see it in the PLS-DA score plot.

-line 213 does not make much sense to me: ”Main differences in the sphingolipidomes were found in the classes of SLs: Cer, SM, Sph, sphinganines, and S1P.” The listed classes are all the classes of SLs. Delete this sentence and focus on telling the readers which SL species (not classes) contributed the  most to the difference among the sample groups.

-line 215: We demonstrated qualitative higher Cer,...ddi you mean quantitative higher? Qualitative analysis is detecting the lipid species in the sample (list of species), and quantitative analysis gives you pmol/mL for each analyte. Qualitative does not tell you higher or lower, just there is/there is not.

-line 215: when I dont see that you summed total ceramide levels or total SM levels.

What do you mean by: ”compared to the others”? All other groups or specific groups?
Also higher or lower is decided by unistat (p-values), VIP values and fold change, as you presented in the Figure 2, so you should save the discussion of difference among groups for later. Heatmaps are just meant to show all the results as a composite picture, for readers to get an overview of data, not for describing higher/lower withotu proper metrics.

-line 234: please replace the word ”modulation” (with altered/changed) unless you have a system that can be finely modulated by adjusting multiple levels. Lipid species are either higher or lower in treated/stimulated groups compared to controls.

-line 238: degree (or extent) of concentration change (not modulation)

 

Please use standard lipid abbreviations as accepted in the lipiddomic community, as in: LIPID MAPS

I cannot tell from your text which ceramide is C24 and which is C24:1. Is the double bond from the fatty acyl residue or from the sphingosine (d18:1).

When comparing MφV+FXT (10⁻⁶ M) with MφV group, both virus-stimulated but only one FTX-rescued, there was an increase in C18 SM and C20 SM species. Please add a comment for decreased matching Cer species: C18 Cer and C20 Cer, which are enzymatically connected to SMs. Sphinomyelin sinthase activation would raise levels of SMs, at the expense of lowering Cer levels. Blockage of sphingomyelinase would augment this effect, maintaining higher SMs and preventing generation of Cer from SMs.

English grammar in lines 250-253 prevented me from picking up the meaning, for example: ”effects direct these enzyme products,”. Do you mean affect directly or something else? Also, is it a preservation when the levels of SM increased? I am confused as the authors did not properly comment on Figures 2A and B, telling us first how the virus altered SL species.

-line 252: the sentence is awakward due to poor grammar. ”Indeed, the notable S1P increase following FXT treatment, these findings support an anti-apoptotic and anti-inflammatory environment.” It is unsustainable to draw such general conclusion on anti-apoptotic and anti-inflammatory environment based solely on S1P changes.

-line 268: How come you mentioned Figure 4, and skipped Figure 3?

-line 268: Grammar; revealed...reduced

-Figures 3 and 4 are all mixed up in subsections 3.3. and 3.4. The text in the subsections does not match the actual Figure numeration.

-line 297: what does it mean: ”at macrophage basal level?”

-line 299: it would be nice if the authors used biological language insetad of groups names. For example, a reader would be more happy to read that: ”MMP levels were lower in the SARS-CoV -stimulated macrophages than in control groups” instead of reading how: ”MMP levels were lower in the MφV groups compared to Mφ groups”. Which are the latter, Mφ groups? I am confused since MφV and Mφ0 could belong to Mφ groups.

What is statistical reduction? Statistuically significant reduction or?
Please use biological language whenever possible, replace ”MφV+FXT compared to MφV” by FXT-exposed and virus-treated versus virus-treated without FXT exposure.

- line 302: ”Evaluating other inflammatory stimulation, such as LPS (1 µg/mL)”.Why did authors not mention LPS exposure in Methods?

-line 303: please correct the expression: ”modulate”, as the meaning of modulate is not increase/decrease or augment/diminish, or highten/lessen but varying the strength, exerting a controling influence on. Hormoen levels are modulated by other hormones. Write: ” ...only IL-6 levels were significantly reduced... whereas IL-1β levels showed a lower trend ...”

-line 304: Whats is ”the treated group” in this context when you have both FXT and LPS treatement?

-line 307: too generalized conclusion, not sustained. You cannot say that ”attenuating the production of inflammatory mediators induced by different pathogenic stimuli.” Just based on one stat. significant change in IL-6 and one pathogenic stimulus (LPS).

-line 353: should be: ”ASM associates with lipid raft microdomains at the cell membrane”. It is a smaller thing that associates with bigger, not vice versa.

Author Response

Response to Reviewer 2 Comments:

Comments and Suggestions for Authors

“Fluoxetine reshapes macrophage membrane sphingolipids and infmammatory responses without affecting extracellular vesicle biogenesis upon inactivated-SARS-CoV-2 stimulation” by J. C.S de Carvalho and co-authors aimed at exploring whether FXT can re-shape sphingolipidome of macrophages by inhibiting ASM-Cer axis. They also measure levels of two Ils and two MMPs, and size distribution of Evs from maxcrophages. While a sound experimental work lies behind the study, it has several serious issues that must be addressed.

Response: Thank you for your positive feedback and for recognizing the novelty and significance of our study.

1. The authors may want to play down the hype on modulation of sphingolipidome, when they presented only eleven ceramides, ten SMs, 4 sphingoid bases and S1P. In the big lipidomic era when we whitness big hundreds of lipids and few dozens of SL species, deciphered at the level of known base + known FA residue, the study before me cannot draw significant conclusions on alterations in SL metabolism based on their results.

 

Response: We acknowledge that our study does not aim to provide a comprehensive, untargeted sphingolipidomic coverage encompassing hundreds of lipid species. Instead, our work was deliberately designed as a targeted, hypothesis-driven lipidomic investigation focused on the accurate identification and quantification of key sphingolipid species directly involved in aSMase-mediated signaling and inflammatory responses. Our analytical strategy prioritized high-confidence molecular annotation, including defined sphingoid bases and fatty acyl residues, robust quantification, and validated fragmentation patterns, rather than the detection of large numbers of low-abundance or ambiguously annotated features. This approach minimizes overannotation and false-positive assignments, which remain important challenges in large-scale untargeted lipidomics. In THP-1-derived macrophages, we consistently detected and quantified a focused panel of ceramides, sphingomyelins, sphingoid bases, and S1P species with well-established roles in membrane organization and aSMase-dependent pathways. These molecular species are directly relevant to the biological questions addressed in this study and provide mechanistic support for our conclusions regarding SARS-CoV-2-induced modulation of sphingolipid metabolism and its reversal by FXT treatment.

We agree that broader lipidomic surveys in other experimental systems may reveal a wider spectrum of sphingolipid species. However, in the context of our cellular model and experimental objectives, the targeted approach employed here provides precise, reproducible, and biologically interpretable data that enable meaningful insights into sphingolipid signaling dynamics. To avoid any potential overstatement, we have also revised the manuscript to more clearly emphasize the targeted nature of our lipidomic analysis and to frame our conclusions accordingly.

 

2. I would certainly not support the expression: ”we provide mechanistic evidence that FXT-induced remodeling of the Cer pathway” without the proper explanation of ceramide pathway (whatever it may be).

 

Response: Our study primarily evaluates changes in selected ceramide species and related sphingolipids in response to SARS-CoV-2 stimulation and FXT treatment, rather than providing a complete functional dissection of the ceramide metabolic pathway. In response to this concern, we have revised the relevant sections of the manuscript to remove this expression and to adopt more accurate and cautious terminology. The revised text now emphasizes that our data indicate modulation of specific ceramide-related species and ASM-associated pathways, without implying a full mechanistic elucidation of ceramide metabolism. Furthermore, we have expanded the results section by a new figure (Figure 3) to clarify our enzymatic, fluxomic, and functional analyses.

 

3. ”The observed increase in SM and S1P levels, accompanied by reduced Cer species, is consistent with pharmacological inhibition of ASM activity.” It may be, but it also may be due to an increased activity of sphingomyelin synthase, enzyme that converts Cer to SM.

 

Response: We agree that, in principle, the observed increase in SM and S1P levels accompanied by reduced ceramide species could also be influenced by enhanced sphingomyelin synthase activity. However, our primary interpretation is strongly supported by extensive literature demonstrating that FXT acts as a functional inhibitor of aSMase, leading to reduced ceramide generation and consequent remodeling of sphingolipid profiles. Accordingly, the lipid alterations observed in our study are fully consistent with the well-established pharmacological effects of FXT on aSMase-dependent pathways. Within the scope of our experimental design, aSMase inhibition represents the most parsimonious and biologically supported explanation for the observed metabolic pattern.

We acknowledge that potential compensatory or auxiliary pathways, including increased sphingomyelin synthase activity or broader metabolic rewiring, cannot be completely excluded. However, rigorous evaluation of such mechanisms would require a dedicated metabolic fluxomics approach, integrating isotope tracing and dynamic pathway analysis, which falls beyond the scope of the present study. These revisions ensure a balanced yet evidence-based interpretation of our findings.

4. The authors omitted to explain how they link increase in S1P with decrease in Cer and increase in SM. They write about Cer as it is a single lipid entity, when it is known that, while one Cer species may increase, the other may decrease. They should perform pathway enrichment analysis and try to link the matching species, suhc as how decreased levels of C18 Cer matches increased levels of C18 SM, and do the same for C20 Cer and C20 SM, C24 Cer and C24 SM, etc. This way, functional enrichment analysis would recognize patterns in altered enzyme activities, since, for example, ASM cannot change sphingoid base or FA residue in SM when converting it into Cer, it can only cleave phosphocholine from SM. In other words, ASM cannot produce C14 Cer from C16 or C18 SM.

 

Response: We thank the reviewer for this highly relevant and technically insightful comment. We fully agree and as correctly noted, enzymatic interconversions within the sphingolipid network preserve both the sphingoid base and fatty acyl chain composition, and therefore direct precursor–product relationships can only occur between matching molecular species (e.g., C18 Cer and C18 SM). To this concern, we have performed a detailed species-matched analysis and incorporated new graphical representations in the revised manuscript (new Figure 3). This figure directly compares individual ceramide and SM species with identical fatty acyl chain lengths and sphingoid bases, allowing the evaluation of coordinated changes across the pathway. This analysis reveals consistent inverse relationships between specific ceramide and SM species (e.g., C18, C20, and C24 species), supporting coordinated remodeling along aSMase-related metabolic routes rather than nonspecific global lipid fluctuations. These results strengthen the mechanistic link between altered S1P, Cer, and SM levels observed in our study. These additions and revisions substantially improve the mechanistic interpretation of sphingolipid remodeling in our experimental system.

 

5. The Discussion is oversimplified, reflecting the lack of lipidomic expertise. For example: ”SM participates in multiple cellular processes, including cell division...”. The authors talk about SM as if it is a single lipid species with uniform role, while there are hundreds of SM species and some could have opposing roles, e.g pro-inflammaytory and anti-inflammatory

 

Response: We agree that, in the original version, certain passages in the Discussion adopted a simplified description of SMs, which may have conveyed the unintended impression of functional uniformity across this highly diverse lipid class. We have substantially revised this section to more accurately reflect the well-established molecular and functional heterogeneity of SM species. We now emphasize that distinct SM species may exert divergent, and in some cases opposing, effects on inflammatory signaling, membrane organization, and cellular responses.

We would like to clarify that this simplification was not due to a lack of lipidomic expertise, but rather reflected an initial effort to maintain accessibility for a broad readership. Our group has extensive experience in SL and lipidomic research, including multiple peer-reviewed publications and long-standing collaborations with internationally recognized leaders in the field, such as Prof. Robert Murphy (University of Colorado - USA). These revisions improve the accuracy and depth of the Discussion while preserving its clarity for non-specialist readers.

 

6. How can you be sure that only acid sphingomyelinase is involved in Cer drop and SM rise? Are there any other Smases at the membrane? Would they be able to interact with FXT? Can you try an assay which would demonsrate the binding:

 

Response: Based on the lipid readouts alone (Cer decrease and SM increase), we cannot unequivocally conclude that only aSMase is involved. Multiple SMases exist and may contribute to sphingolipid remodeling depending on cellular context. Nevertheless, our focus on aSMase is supported by the known mechanism of action of FIASMAs such as FTX, which pharmacology act as functional inhibitors of aSMase through lysosomotropic accumulation rather than direct enzymatic binding [1]. In addition, studies by Carpinteiro et al. demonstrated that SARS-CoV-2–induced ceramide platform formation at the plasma membrane is aSMase-dependent and can be disrupted by FIASMAs, providing a mechanistic rationale for implicating aSMase in this context [2,3]. nSMases are also present in membrane-associated compartments and primarily linked to exosome biogenesis. In our study, nSMase activity was functionally addressed using GW4869 in EV assays; however, FTX did not reproduce the effects of nSMase inhibition on EV release. We have revised the manuscript to clarify these points and to avoid overstatement regarding exclusive involvement of aSMase.

 

[1] Le Corre P, Loas G. Repurposing functional inhibitors of acid sphingomyelinase (fiasmas): an opportunity against SARS-CoV-2 infection? J Clin Pharm Ther [Internet]. 2021;46(5):1213–9. Available from: https://doi.org/10.1111/jcpt.13390;

[2] Carpinteiro A, Gripp B, Hoffmann M, Pöhlmann S, Hoertel N, Edwards MJ, et al. Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells. J Biol Chem [Internet]. 2021;296:100701. Available from: https://doi.org/10.1016/j.jbc.2021.100701

[3] Carpinteiro A, Edwards MJ, Hoffmann M, Kochs G, Gripp B, Weigang S, et al. Pharmacological Inhibition of Acid Sphingomyelinase Prevents Uptake of SARS-CoV-2 by Epithelial Cells. Cell Reports Med [Internet]. 2020;1(8). Available from: https://doi.org/10.1016/j.xcrm.2020.100142

 

7. Also want to see PCA plots.

Response: In response, we have now performed PCA on the lipidomic dataset and included the corresponding score plots in the revised manuscript as Supplementary Figure 2. The PCA results support the consistency and robustness of our measurements and further corroborate the treatment-dependent remodeling of the sphingolipidome observed in our targeted analysis.

 

8. The lack of expertise in lipid species, their nature, nomenclature and enzymatic conversions is obvious. I want to see  clear comments on alterations in Cer and SM; levels in Figures 2A, 2B and 2C, comparing stimulated and non-stimulated groups, FXT-treated vs non-treated etc. The authors gave only two vague sentences, when combined with poor grammar and generalized conclusion (which belong to the Discussion) I learnt nothing. It is not readers job to analyse the Figures and draw conclusions.

Response: We acknowledge that, in the previous version of the manuscript, the interpretation of Cer and SM alterations in Figures 2A–C could be improved in terms of clarity and grammatical precision. We agree that it is the authors’ responsibility to guide the reader through the data and to provide a clear, biologically meaningful interpretation. We have implemented substantial revisions to both the Results and Discussion sections. We have added a new dedicated subsection entitled “Indirect pathway enrichment analysis reveals fluoxetine-sensitive sphingomyelinase activity in macrophages”, in which we now provide a detailed, systematic, and species-resolved interpretation of Cer and SM alterations across stimulated versus non-stimulated conditions and FXT-treated versus non-treated macrophages. This revised section explicitly describes coordinated changes in matched Cer and SM species and their modulation by SARS-CoV-2 stimulation and FXT treatment. The analysis highlights patterns consistent with altered SMase-dependent remodeling, supported by quantitative comparisons and pathway-oriented interpretation. In particular, the data demonstrate differential SMase-related activity in macrophages treated with 10⁻⁶ M FXT, consistent with regulated SM–Cer interconversion.

Furthermore, we have carefully revised the text for grammatical accuracy and scientific precision and relocated generalized interpretative statements to the Discussion, ensuring a clear separation between results presentation and broader interpretation. We would also like to clarify that our research group has extensive experience in lipidomics and lipid biology, supported by a substantial publication record in the field. The issues identified here reflected limitations in the initial presentation rather than a lack of technical expertise.

 

9. Authors tend to use generalized conclusions (hypotheses) not really based on data obtained, such as: ”Indeed, the notable S1P increase following FXT treatment, these findings support an anti-apoptotic and anti-inflammatory environment.”

 

Response: We have revised the relevant sections of the manuscript to remove generalized or hypothesis-driven conclusions that are not directly substantiated by our experimental results. The revised text now presents the observed changes in S1P levels in a descriptive and evidence-based manner, avoiding functional inferences in the absence of direct apoptosis or inflammatory signaling assays. Furthermore, we have carefully reviewed the entire Discussion and Conclusion sections to ensure that all interpretations are firmly grounded in the presented data and appropriately framed as preliminary where necessary.

 

10. Much more experiments should be done in order to conclude this. Also, ”The TEM analyses revealed that treatment with FXT substantially reduces the average diameter of EVs released after stimulation with SARS-CoV-2 particles, indicating that FXT treatment modulates cargo composition and diminishes the EVs size.” EV size and size distribution was analysed on TEM, but, cargo composition was not.

 

Response: We agree that this statement was too speculative in the context of the data presented. The text has been revised to remove generalized conclusions and to ensure that interpretations are strictly supported by the experimental results.

 

11. line 307: One cannot conclude that ”attenuating the production of inflammatory mediators induced by different pathogenic stimuli” simply based on one stat. significant change in IL-6 and one pathogenic stimulus (LPS).

 

Response: We have revised the corresponding section of the manuscript to avoid generalization beyond the experimental evidence. The revised text now specifically refers to the observed modulation of IL-6 under LPS stimulation, without extrapolating to other inflammatory mediators or pathogenic stimuli. 

12. The manuscript leved an impression that it was hastily written, without a critical deep reading. I noticed that authors commented on Figures 4 in 3.3, but the plots and pictures were in Figure 3. This is not a sole example. In the subsection 3.4. the authors discuss Figure 4, again.

 

Response: We made the necessary corrections to the body of the text, citing the figures into the correct fields.

13. The experiment with LPS treatment of macrophages was not even mentioned in Methods.

 

Response:  We have revised this point accordingly in the methods section.

14. I like the picture 4I.

 

Response: We thank the reviewer for this positive comment and are pleased that Figure 4I was found to be informative. We appreciate this feedback.

Other issues:

15. line 55: not only in macrophages are lipid rafts enriched in cholesterol…

 

Response: We agree that lipid rafts are enriched in cholesterol and sphingolipids in multiple cell types, not exclusively in macrophages. In response, we have revised the corresponding sentence

16. line 61: not all SLs are bioactive  lipids, actually most of them are structural components of cell membranes, especially sphingomyelins. Biaoctive lipids are sphingoid bases (S1P), ceramides, ceramide-1-phosphate (Cer1P) and several others.

 

Response: Accordingly, we have revised the corresponding statement in line 61 to accurately reflect this distinction between structural and signaling sphingolipids, thereby improving the conceptual clarity of the Introduction.

17. line 65: De novo SLs bio..

 

Response: This revision improves the conceptual accuracy and clarity of the Introduction.

18. line 68-70: Why are authors talking about ceramide in singular? It should be ceramides (as well as sphingomyelins), since many dozens of different fatty acyl residues (FA) can be attached to sphinganines (giving rise to dhCer) or sphingosines (giving rise to ceramides), not to mention sphingoid bases with different number of C-atoms such as C16-sphingosine, C18 sphingosine, C19 sphingosine, C20 sphingosine, etc.

 

Response: Thank you for your feedback; we have made the necessary corrections to this paragraph.

19. line 69: HexCer is glycosyl ceramide, since Hex can be Glc or Gal. If you are sure that it was glucose, the name is glucosyl ceramide (GlcCer), and if you are sure that it was galactose, the name is galactosyl ceramide (GalCer). When not sure about the nature of the hexose, then, the name is hexosyl ceramide (HexCer).

 

Response: Accordingly, we have revised the manuscript to consistently use the term “HexCer” in cases where the nature of the hexose residue could not be definitively assigned. This correction ensures accurate and standardized lipid nomenclature throughout the manuscript.

 

20. line 70: LacCer is lactosyl-ceramide (Hex2Cer), since Lac consist of one Glc and one Gal.

 

Response: We have corrected the manuscript to specify this issue.

21. line 71: many lipid species can be considered as key signaling lipids, so let us change this into: S1P, a key lipid involved in immune cell functions.

 

Response: We made the necessary corrections to the text.

22. line 77: which Cer species promotes the listed functions? It is written in singular, so I cannot tell which one? Let em give you an examples, C16-Cer has a pro-inflammatory effect promoting insulin resistance, activvates NF-kappaB, thus controbuting to metabolic syndrome, whereas C24-Cer suppresses inflammatory signaling and protects against tissue damage, correlating with reduced risk of cardiovascular disesase. In the light of these facts, one cannot simply put dozens of different CERAMIDES into one lipid with one function.

 

Response: We agree that ceramide species with different acyl-chain lengths can exert distinct, and sometimes opposing, biological effects. Indeed, specific ceramide species such as C16-Cer and C24-Cer have been reported to differentially regulate inflammatory and metabolic signaling pathways. In our analysis, ceramide species were quantified separately, and some species with distinct chain lengths were modulated by FXT treatment. However, we acknowledge that in the original manuscript, we used the term "ceramides" in a simplified way, which may suggest a uniform biological function. To address this issue, we have revised the text to emphasize the heterogeneous nature of ceramide species and to clarify that the observed functional effects are interpreted in the context of coordinated changes across multiple ceramide subspecies rather than ascribing a single function to the entire ceramide class.

23. line 80: please inform us first what is LPS; then use the abbreviation.

 

Response: We made the necessary corrections to the text.

24. line 81: I wish the authors could be a bit more specific when writing about: ”with discrete metabolites correlating with distinct activation states [8].”

 

Response: The revised text now specifies representative sphingolipid species and metabolic patterns associated with different macrophage activation states, thereby strengthening the conceptual link between lipid metabolism and functional phenotypes.

25. Which are these discrete metabolites?ž--line 87 is a bit wordy, I could not understand what is promoter of what? Do ceramides, produced from SMs by SMases, drive or promote driving? What do you mean by: ”driving of intraluminal vesicles withing MVB?”

 

Response: We have substantially revised this sentence to clearly identify the relevant sphingolipid species and to explicitly describe their proposed functions, facilitating intraluminal vesicle budding within MVBs.

26. Line 89: please use a different term from the ”lipid vesicles”, since they are membranous structures but also carry non-lipid cargo or?

 

Response: We have revised the text to replace the term “lipid vesicles” with more appropriate terminology.

27. line 93: what is the difference between EXTRACELLULAR and intercellular microenviroment? They are both outside the cell. Also, the expression: ”supporting the physiological processes” is so wide, as literally everything occurring inside the body is covered by this expression. Please be more specific when writing about the roles of Evs. Which processes?

 

Response: We have revised the corresponding sentence to eliminate this redundancy and to provide a more specific and mechanistically meaningful description of extracellular vesicle functions. The revised text now emphasizes the role of EVs in cell-to-cell communication through the transfer of bioactive molecules and specifies their involvement in immune regulation, inflammatory signaling, tissue homeostasis, and cellular stress responses.

28. line 96: please writeh what is ESCRT pathway.

 

Response: We agree that the ESCRT pathway was not sufficiently defined in the original manuscript. In response, we have revised the corresponding section to explicitly define ESCRT as the endosomal sorting complex required for transport and to briefly describe its role in multivesicular body formation and extracellular vesicle biogenesis: “Additionally, the Cer transfer protein CERT is directly involved in regulating both the Cer and SM content of EVs, thus linking SLs metabolism to EV biogenesis through the ESCRT (endosomal sorting complex required for transport) pathway (16-19) (also include references: https://pubmed.ncbi.nlm.nih.gov/24140720/; https://onlinelibrary.wiley.com/doi/10.1002/jnr.23798; https://pubmed.ncbi.nlm.nih.gov/18309083/)”

29. Line 100: you can remove it, as you have already mention changes in SLs composition in line 97, and since roles in neurodegeneration, cardiovascular disease, and cancer progression are not in your line of research presented in this manuscript. The introduction is too long. You may want to stick to inflammation caused by viral diseases.

Response: We agree that this information was redundant and not directly aligned with the main focus of the present study., then we removed it.

30. line 104: please delete : ”an enzyme responsible for Cer production”, it is a repetition (line 71). Also it may sound ambiguous in this context, since there are other enzymes that produce Cer.

 

Response: We agree with the reviewer's concern and have corrected the points mentioned.

31. Line 106, please explain the abbreviation FIASMA.

 

Response: We made the necessary corrections to the text.

32. line 107: please check the grammar...”such as diminishes”, should be: ...”such as diminishing Cer-rich membrane domains”.

 

Response: We made the necessary corrections to the text.

33. line 110: chronic FXT treatment of what? Which cells?

 

Response: We made the corrections and addressed the necessary details.

 

34. line 113: should be: ”...and, potentially, susceptibility of cells to viral infection”.

 

Response: We made the necessary corrections to the text.

35. line 114-116: be aware of the grammar, when you mention FXT along with SARS-CoV-2, then it is vague which of the two is under: ”ITS broader immunomodulatory…”.

 

Response: We made the necessary corrections to the text.

36. line 117: be aware of the grammar: ...inactivated particles induces...(plural).

 

Response: We made the necessary corrections to the text.

37. line 118: it would be nice to know which are these ”key pro-inflammatory mediators commonly elevated in COVID-19”.

 

Response: We made the necessary corrections to the text.

38. line 119, please be more specific, tell us which cellular membranes are mentioned in : ”remodels the SLs composition in cellular membranes”.

 

Response: The text has been revised to specify the cellular membranes involved, including the plasma membrane, endosomal membranes, and membranes of multivesicular bodies, which are particularly relevant to extracellular vesicle biogenesis and immune signaling in macrophages.

39. line 120: be aware of the grammar, it is vague what you meant in: ”affected under these conditions”. Which are these conditions? COVID infection or FTX treatment, since both of the two were mentioned in the previous sentence.

 

Response: We made the necessary corrections to the text.

40. line 122: please change into: ” …we provide new evidence of FTX repurposing in regulating”. Delete the word: ”collectively”, as I don’t understand what is here collectively, all of the authors together doing the study or?.

 

Response: We made the necessary corrections to the text.

41. line 123: please explain: ” …membrane lipid metabolism”. Is there specific membrane lipid metabolism apart from the common lipid metabolism, or did you mean ”membrane lipid re-organization”.

 

Response: We made the corrections to means: “remodeling”

42. line 124: please change into: ” …beyond being an anti-depressant, towards”.

 

Response: We made the necessary corrections to the text.

43. line 129:…for the prophylactic exposure….

 

Response: We made the necessary corrections to the text.

 

44. line 130:…following THE treatment…to remove THE residual drug.

 

Response: We made the necessary corrections to the text.

45. line 132: it is very difficult to undersand the study design without the sentence telling us that control differentiated macropohages were not treated with FXT, but later were stimulated by SARS CoV2. I wish you could just simply tell us that you had 4 groups: i) THP-1 macrophages treated with FXT and stimulated by virus (MφV-FXT), ii) THP-1 macrophages not treated with FXT, but stimulated by virus (MφV), iii) THP-1 macrophages not treated with FXT and not stimulated by virus (double-negative control, Mφ0) and iv) THP-1 macrophages treated with FXT but not stimulated by virus (Mφ0-FXT).

 

Response: We made the necessary corrections to the text.

46. line 136:…for THE subsequent EVs isolation…

 

Response: We made the necessary corrections to the text.

 

47. Please use MACROPHAGES insetad of CELLS, wherever possible, to avoid confusion.

 

Response: We made the necessary corrections to the text.

 

48. line 139: please delete the: ”For SLs extraction” and start with: 1×10⁶ macrophage cells were suspended…since the subtitle says it all.

 

Response: We made the necessary corrections to the text.

 

49. We would need the full noun of PBS here.

 

Response: We made the necessary corrections to the text.

50. I need to know why did you add HCL in the extraction solvent (Folch, chloroform/Methanol 2/1)?

 

Response: The addition of HCl to the extraction solvent was performed to promote sample acidification, which facilitates the disruption of protein–lipid and lipid–lipid interactions and improves phase separation during the modified Folch extraction. Acidification also enhances the recovery of acidic and polar lipid species, including sphingolipids and phospholipids, by preventing their retention in the interphase and aqueous phase. Moreover, the acidic environment contributes to minimizing enzymatic activity during extraction, thereby reducing post-sampling lipid degradation and artificial remodeling. This approach has been previously described in modified Folch-based protocols for lipidomics to improve extraction efficiency and reproducibility, particularly for bioactive sphingolipids. We have now clarified this rationale in the Methods section to improve transparency and reproducibility.

51. line 143:…and THE second extraction…please avoid wordiness and use shorter sentences, like this one: ”after the organic phase was collected, we re-extracted the aqueous phase with another 2 mL of CHCl3.”.

 

Response: We made the necessary corrections to the text.

 

52. We perfomed extraction = we extracted.

Response: We made the necessary corrections to the text.

 

53. We perfomed analysis = we analysed etc etc…

 

Response: We made the necessary corrections to the text.

 

54. line 144 should read: ”combined organic phase with the extracted lipids was evaporated…then resuspended in…”(when you combine the two, you get one combined solution).

 

Response: We made the necessary corrections to the text.

55. line 155: ”Lipids were analysed using a TripleTOF 5600+ mass spectrometer”.I corrected this since you did not only DETECT lipid species (qualitative analysis), you quantified them (quantitative analysis=measuring lipid concentrations/abundances).

 

Response: We made the necessary corrections to the text.

56. line 180: all capital letters Extracellular Vesicles Characterization.

 

Response: We made the necessary corrections to the text.

57. line 184:…for THE morphological assessment…

 

Response: We made the necessary corrections to the text.

58. line 188:…visualized using a JEM-2100…

 

Response: We made the necessary corrections to the text.

59. lines 134 and 189, duplication of FMRP-USP. What is it?

 

Response: We made the necessary corrections to the text.

60. line 190: please change the title into Quantification of protein mediators.

 

Response: We made the necessary corrections to the text.

61. line 191: be aware of the grammar: ...secretion of...were quantified (should be singular). Also you measured concentration of cytokines and MMPs and not secretion. Hence, write: We measured cytokines: IL-10....as well as MMPs:

 

Response: We made the necessary corrections to the text.

62. line 199: please be careful of the correct terms. You should write: ”Multivariate analyses were conducted using…”. Lipidomic analysis is when you perform qualitative and quantitative analysis of the lipid species in your samples, and this can be done only by LC MS/MS. Then you use the columns with pmol/mL values for these lipids and input it into software tools such as MetaboAnalystR, ggplot2 and ComplexHeatmap. That is called DATA analysis, and since you have many variables (measure lipids) it is also called multivariate data analysis. Therefore, you cannot perform Multivariate lipidomics analyses using R packages. R packages see data solely as numbers (data is data, regardless their origin).

Response: We have incorporated the necessary corrections into the text.

63. line 201: what do you mean under ”parametric comparisons”? I know lipid species are usually non-normally distributed, did you check for these distributions prior to ANOVA?.

 

Response: By “parametric comparisons,” we refer to the use of parametric statistical tests, specifically one-way or two-way ANOVA followed by appropriate post hoc analyses, to compare lipid species across experimental groups. Prior to performing ANOVA, we evaluated the distribution of the data and the homogeneity of variances using standard normality (Shapiro–Wilk test) and variance homogeneity (Levene’s test) assessments. When the assumptions of normality and homoscedasticity were not met, data were log-transformed to improve distribution symmetry. In cases where these assumptions could not be satisfied, non-parametric alternatives were applied.

 

64. line 207: please shorten into: ” Figure 1A depicts how FXT treatment affected the sphingolipidome in THP-1-derived macrophages. ” It is all sadi in this short sentence. I corrected this because when you write: ”…modulate macrophage lipid profiles”, one expects to see the total lipidome of macrophages, that is phospholipids, glycerolipids etc, not only SLs.

 

Response: We have incorporated the necessary corrections into the text.

65. line 210, please use SL instead of sphingolipid.

 

Response: We agree with the reviewer's concern and have corrected the points mentioned.

66. line 210: I dont understand the meaning of: ” similar overall sphingolipid profiles, despite the intensity of Cer and SM on MφV group”. What is exactly the intensity of Cer and SM in group? All measured ceramides or one specific Cer species (same for SM)?

 

Response: To address this concern, we have generated a new Figure 3 and supplementary figure, which provides a detailed and quantitative description of Cer and SM species. These figures present the normalized abundances of each detected molecular species across experimental groups, allowing a clearer interpretation of the specific changes in Cer and SM profiles. The inclusion of this figure enables a more precise comparison between stimulated and non-stimulated conditions, as well as between FTX-treated and untreated macrophages, thereby strengthening the interpretation of sphingolipid remodeling in our model.

67. Why didnt you acquaint us about the different doses of FTX applied to the studied groups in Methods? It seems all of a suddent to see it in the PLS-DA score plot.

 

Response: We acknowledge that the description of the FXT concentrations used in the experimental groups was insufficiently detailed in the original version of the manuscript. In response, we have now added these details to both the Methods and Results sections, explicitly reporting all FXT doses applied in each experimental condition, including those represented in the PLS-DA score plot.

68. line 213 does not make much sense to me: ”Main differences in the sphingolipidomes were found in the classes of SLs: Cer, SM, Sph, sphinganines, and S1P.” The listed classes are all the classes of SLs. Delete this sentence and focus on telling the readers which SL species (not classes) contributed the  most to the difference among the sample groups.

 

Response: We thank the reviewer for pointing this out. Actually, as our targeted method also includes glycosphingolipids, and we did not detect these species, we made it clear within the text which exact classes were found, not generalizing to lead to a misunderstanding for the readers.

 

69. line 215: We demonstrated qualitative higher Cer,...ddi you mean quantitative higher? Qualitative analysis is detecting the lipid species in the sample (list of species), and quantitative analysis gives you pmol/mL for each analyte. Qualitative does not tell you higher or lower, just there is/there is not.

 

Response: We thank the reviewer for this important clarification. We agree that the term “qualitative” was inappropriate in this context. Our analysis is based on quantitative LC–MS/MS measurements, and we now provide the corresponding numerical data in Supplementary Table 2. Accordingly, we have revised the manuscript to use “quantitative” instead of “qualitative” and to explicitly refer to these results.

70. line 215: when I dont see that you summed total ceramide levels or total SM levels.

 

Response: We have incorporated the necessary corrections into the text.

71. What do you mean by: ”compared to the others”? All other groups or specific groups?
    Also higher or lower is decided by unistat (p-values), VIP values and fold change, as you presented in the Figure 2, so you should save the discussion of difference among groups for later. Heatmaps are just meant to show all the results as a composite picture, for readers to get an overview of data, not for describing higher/lower withotu proper metrics.

 

Response: We fully agree that conclusions regarding higher or lower lipid levels should be based on appropriate statistical analyses, including univariate statistics (p-values), VIP scores, and fold-change values, rather than on heatmap visualization alone. Accordingly, we have revised the text to avoid interpretative statements based solely on heatmaps and now refer explicitly to the quantitative data and statistical metrics presented in Figure 2, Supplementary Figure 2 and Supplementary Table 2. The heatmaps are now described strictly as an overview tool to visualize global patterns in the dataset, while the detailed comparisons among experimental groups are discussed based on validated quantitative and statistical analyses.

 

72. line 234: please replace the word ”modulation” (with altered/changed) unless you have a system that can be finely modulated by adjusting multiple levels. Lipid species are either higher or lower in treated/stimulated groups compared to controls.

 

Response: We have incorporated the necessary corrections into the text.

73. line 238: degree (or extent) of concentration change (not modulation).

 

Response: We have incorporated the necessary corrections into the text.

74. Please use standard lipid abbreviations as accepted in the lipiddomic community, as in: LIPID MAPS.

 

Response: We have incorporated the necessary corrections into the text.

75. I cannot tell from your text which ceramide is C24 and which is C24:1. Is the double bond from the fatty acyl residue or from the sphingosine (d18:1).

 

Response: In our LC–MS/MS workflow, sphingolipid species were identified using a validated fragmentation library that allows discrimination between variations in the fatty acyl chain and the sphingoid base. Based on characteristic fragment ions, we can confirm that the annotation of C24 and C24:1 refers to differences in the fatty acyl residue and not to variations in the sphingoid backbone (which was consistently d18:1 in the detected species).

 

76. When comparing MφV+FXT (10⁻⁶ M) with MφV group, both virus-stimulated but only one FTX-rescued, there was an increase in C18 SM and C20 SM species. Please add a comment for decreased matching Cer species: C18 Cer and C20 Cer, which are enzymatically connected to SMs. Sphinomyelin sinthase activation would raise levels of SMs, at the expense of lowering Cer levels. Blockage of sphingomyelinase would augment this effect, maintaining higher SMs and preventing generation of Cer from SMs.

 

Response: In the revised manuscript, this relationship is now explicitly addressed in the Results and Discussion sections and supported by the newly included Figure 3. We now describe that, in MφV+FXT (10⁻⁶ M) cells, the increase in C18 and C20 SM is accompanied by a reduction in the corresponding ceramide species, consistent with reduced sphingomyelinase-mediated hydrolysis and/or enhanced sphingomyelin synthase activity. Furthermore, we emphasize that pharmacological inhibition of acid sphingomyelinase by FXT, as supported by previous literature, is expected to limit ceramide generation from sphingomyelins, thereby maintaining elevated SM levels and preventing accumulation of matching ceramide species. We also acknowledge that a definitive distinction between sphingomyelinase inhibition and compensatory activation of sphingomyelin synthases would require dedicated metabolic flux analyses, which are beyond the scope of the present study and represent an important direction for future investigations.

 

77. English grammar in lines 250-253 prevented me from picking up the meaning, for example: ”effects direct these enzyme products,”. Do you mean affect directly or something else? Also, is it a preservation when the levels of SM increased? I am confused as the authors did not properly comment on Figures 2A and B, telling us first how the virus altered SL species.

 

Response: We have incorporated the necessary corrections into the text.

78. line 252: the sentence is awakward due to poor grammar. ”Indeed, the notable S1P increase following FXT treatment, these findings support an anti-apoptotic and anti-inflammatory environment.” It is unsustainable to draw such general conclusion on anti-apoptotic and anti-inflammatory environment based solely on S1P changes.

 

Response: The paragraph was replaced with: “Indeed, the notable increase in S1P after FXT treatment, in combination with the observed changes in ceramide and inflammatory mediator levels, may contribute to a less inflammatory cellular state and reduced susceptibility to cellular stress.”

79. line 268: How come you mentioned Figure 4, and skipped Figure 3?

 

Response: We have incorporated the necessary corrections into the text.

 

80. line 268: Grammar; revealed...reduced.

 

Response: We have incorporated the necessary corrections into the text.

81. Figures 3 and 4 are all mixed up in subsections 3.3. and 3.4. The text in the subsections does not match the actual Figure numeration.

 

Response: We have incorporated the necessary corrections into the text. 

82. line 297: what does it mean: ”at macrophage basal level?”

 

Response: The phrase “at macrophage basal level” was intended to refer to untreated, non-stimulated macrophages, representing their resting or homeostatic state prior to any viral or pharmacological stimulation. To avoid ambiguity, we have revised the text to explicitly state “in resting (non-stimulated) macrophages” throughout the manuscript.

83. line 299: it would be nice if the authors used biological language insetad of groups names. For example, a reader would be more happy to read that: ”MMP levels were lower in the SARS-CoV -stimulated macrophages than in control groups” instead of reading how: ”MMP levels were lower in the MφV groups compared to Mφ groups”. Which are the latter, Mφ groups? I am confused since MφV and Mφ0 could belong to Mφ groups.

 

Response: We have incorporated the necessary corrections into the text.  

84. What is statistical reduction? Statistuically significant reduction or?
    Please use biological language whenever possible, replace ”MφV+FXT compared to MφV” by FXT-exposed and virus-treated versus virus-treated without FXT exposure.

 

Response: We have incorporated the necessary corrections into the text.

85. line 302: ”Evaluating other inflammatory stimulation, such as LPS (1 µg/mL)”.Why did authors not mention LPS exposure in Methods?

 

Response: We have incorporated the necessary corrections into the Methods section.

 

86. line 303: please correct the expression: ”modulate”, as the meaning of modulate is not increase/decrease or augment/diminish, or highten/lessen but varying the strength, exerting a controling influence on. Hormoen levels are modulated by other hormones. Write: ” ...only IL-6 levels were significantly reduced... whereas IL-1β levels showed a lower trend ...”

 

Response: We have incorporated the necessary corrections into the text.

87. line 304: Whats is ”the treated group” in this context when you have both FXT and LPS treatement?

 

Response: We have incorporated the necessary corrections into the text.

88. line 307: too generalized conclusion, not sustained. You cannot say that ”attenuating the production of inflammatory mediators induced by different pathogenic stimuli.” Just based on one stat. significant change in IL-6 and one pathogenic stimulus (LPS).

 

Response: We have incorporated the necessary corrections into the text.

89. line 353: should be: ”ASM associates with lipid raft microdomains at the cell membrane”. It is a smaller thing that associates with bigger, not vice versa.

Response: We agree that, mechanistically, aSMase associates with pre-existing lipid raft microdomains rather than lipid rafts associating with aSMase. Accordingly, we have revised the text to reflect this correct relationship. The sentence now states that aSMase associates with cholesterol- and sphingolipid-enriched lipid raft microdomains at the plasma membrane, where its recruitment promotes local Cer generation, lipid raft clustering, and the amplification of raft-dependent signaling processes, as supported by previous reports (PMID: 18772496; 32328634; S0021925825020629). This revision improves the conceptual accuracy of the manuscript and aligns our interpretation with established literature.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has demonstrated significant improvement​ in overall quality. While several minor issues remain to be discussed with the authors to further enhance clarity and rigor.

1. The labeling of “acid sphingomyelinase (aSMase)”in Figure 5 should be revised. The figure 5 have multiple issues, including ambiguous directionality of extracellular vesicles (EVs) and the distribution of sphingomyelin and ceramide across intracellular/extracellular compartments.

2. The relationship between aSMase activity, EV directionality, and SM/Cer dynamics raises a question: compare the ratios and directional changes of Cer to SM​ in cell culture media versus intracellular vesicles. This comparison would clarify whether Cer accumulation/shedding differs between extracellular and intracellular pools.

1. The proposed mechanism by which FTX inhibits aSMase and alters EV size remains unclear. Following viral binding to the ACE2 receptor, how does SM clustering recruit endocytic machinery to form endosomes that activate aSMase? Are alternative pathways (e.g., direct aSMase activation at the plasma membrane) involved? The link between aSMase inhibition by FTX and EV size modulation requires elaboration. For example, does aSMase inhibition alter lipid raft composition, thereby affecting EV budding or cargo sorting? The authors elaborate on these mechanistic points in the Discussion section​ to strengthen the interpretability of their findings, particularly regarding the specificity of aSMase inhibition and its impact on EV biology.

Comments on the Quality of English Language

The phrase “cells were under these conditions :”is grammatically awkward. It is recommended to revise it to: “The cells were cultured under these conditions”. Please check through the text.

Author Response

Comments: The manuscript has demonstrated significant improvement​ in overall quality. While several minor issues remain to be discussed with the authors to further enhance clarity and rigor.

1. The labeling of “acid sphingomyelinase (aSMase)”in Figure 5 should be revised. The figure 5 have multiple issues, including ambiguous directionality of extracellular vesicles (EVs) and the distribution of sphingomyelin and ceramide across intracellular/extracellular compartments.

Response: We thank the reviewer for this important and constructive comment. We agree that the original version of Figure 5 could lead to ambiguity regarding the labeling of acid sphingomyelinase (aSMase), the directionality of extracellular vesicle (EV) trafficking, and the compartmental distribution of sphingomyelin and ceramide. To address these concerns, Figure 5 has been carefully revised. Specifically: (i) the labeling of aSMase was corrected and repositioned to clearly indicate its site of action; (ii) the directionality of EV biogenesis and release has been clarified by adding explicit arrows and compartment boundaries; and (iii) the localization of SM and Cer was refined to accurately reflect their intracellular and extracellular distributions, in line with current knowledge of SL metabolism and EV biology. These modifications improve the clarity and accuracy of the schematic representation and reduce the possibility of misinterpretation

2. The relationship between aSMase activity, EV directionality, and SM/Cer dynamics raises a question: compare the ratios and directional changes of Cer to SM​ in cell culture media versus intracellular vesicles. This comparison would clarify whether Cer accumulation/shedding differs between extracellular and intracellular pools.

Response: In the present study, SL profiling was performed on whole-cell extracts, allowing us to assess relative changes in SM and Cer associated with membranes and EV release. However, a direct quantitative comparison of Cer/SM ratios between total cell culture media and distinct intracellular vesicular compartments (e.g., endosomes or multivesicular bodies) was beyond the experimental scope of this work. Importantly, our data indicate that FXT treatment leads to intracellular accumulation of SM concomitant with reduced Cer generation, consistent with inhibition of aSMase activity. In EVs, although total vesicle release remained unchanged, we observed alterations in vesicle size distribution, suggesting changes in the cargo. This supports the interpretation that Cer accumulation is preferentially retained within intracellular membranes rather than being exported under FXT treatment. Future studies employing subcellular fractionation or targeted lipid flux analyses will be required to directly quantify Cer/SM ratios across specific intracellular vesicular compartments and the extracellular environment.

3. The proposed mechanism by which FTX inhibits aSMase and alters EV size remains unclear. Following viral binding to the ACE2 receptor, how does SM clustering recruit endocytic machinery to form endosomes that activate aSMase? Are alternative pathways (e.g., direct aSMase activation at the plasma membrane) involved? The link between aSMase inhibition by FTX and EV size modulation requires elaboration. For example, does aSMase inhibition alter lipid raft composition, thereby affecting EV budding or cargo sorting? The authors elaborate on these mechanistic points in the Discussion section​ to strengthen the interpretability of their findings, particularly regarding the specificity of aSMase inhibition and its impact on EV biology.

Response: FXT belongs to the class of FIASMAs, whose pharmacological action is confined to acidic intracellular compartments, particularly late endosomes and lysosomes. Due to their weakly basic and lipophilic nature, FIASMAs passively diffuse across membranes and become protonated and trapped within acidic organelles, where they induce the detachment and subsequent proteolytic degradation of aSMase. Importantly, this mechanism precludes direct inhibition of aSMase at the plasma membrane, since it does not work as a competitive enzyme inhibitor, binding to the active site.

In this context, the initial steps of viral engagement with the ACE2 receptor and the associated clustering of SM-rich lipid rafts at the plasma membrane are unlikely to be directly affected by FXT. Instead, SM clustering facilitates receptor internalization and the formation of early endocytic vesicles, which subsequently mature into late endosomes. During endosomal maturation, luminal acidification promotes optimal aSMase activity, leading to SM hydrolysis and Cer accumulation within the endosomal membrane. FXT-mediated inhibition of aSMase therefore occurs downstream of viral entry, specifically at the level of late endosomes. By preventing Cer generation in these compartments, FXT disrupts Cer-driven membrane reorganization, which is essential for endosomal maturation, intraluminal vesicle formation, and ESCRT-independent budding processes. As Cer plays a critical role in promoting negative membrane curvature and vesicle scission, its depletion is expected to alter the biophysical properties of endosomal membranes. These alterations provide a mechanistic basis for the observed modulation of EV size. Reduced Cer availability within endosomal membranes may impair membrane constriction during vesicle budding, favoring the release of larger EVs and altering EV heterogeneity. Additionally, changes in endosomal lipid composition may affect cargo sorting into EVs, further contributing to shifts in EV size distribution.

Thus, the effect of FXT on EV biology is best explained not by inhibition of plasma membrane–associated aSMase activity, but by selective disruption of endosomal Cer generation. This endosome-restricted mechanism is fully consistent with the known pharmacology of FIASMAs and supports a model in which FXT indirectly modulates EV biogenesis and size through its impact on SL metabolism within acidic intracellular compartments. However, the precise molecular intermediates remain to be elucidated. We therefore frame our conclusions cautiously and highlight this pathway as an important target for future mechanistic studies, for now, in this paper, it is essential to demonstrate the biological effect within immunometabolism scenarium. 

Minor: The phrase “cells were under these conditions :”is grammatically awkward. It is recommended to revise it to: “The cells were cultured under these conditions”. Please check through the text.

Response: We thank the reviewer for pointing out this grammatical issue. The phrase has been revised. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors made an extensive effort to improve the original version of their manuscript. It is now scientifically sound, with a clear presentation of the results and a more concise and relevant discussion. The authors convinced me that they possess a lipidomics expertise, therefore, I will recommend this manuscript for publication in Membranes.

Author Response

Comments: The authors made an extensive effort to improve the original version of their manuscript. It is now scientifically sound, with a clear presentation of the results and a more concise and relevant discussion. The authors convinced me that they possess a lipidomics expertise, therefore, I will recommend this manuscript for publication in IJMS.

Response: We sincerely thank Reviewer 2 for this very positive and encouraging assessment. We are grateful for the recognition of our efforts to improve the manuscript, as well as for the acknowledgment of the scientific soundness, clarity of data presentation, and the relevance of the revised discussion. We particularly appreciate the reviewer’s confidence in our lipidomics expertise and their recommendation for publication in this journal. These comments were highly motivating and valuable for us.