ArtinM Modulates Intestinal Inflammation in Acute Experimental Trypanosoma cruzi Infection with External Single-Cell Transcriptomic Contextualization

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Congratulations for the original, sophisticated and original research program.

Author Response

Reviewer 1

Reviewer comment:

Congratulations for the original, sophisticated and original research program.

Response:
We sincerely thank the reviewer for this generous and encouraging comment. We are pleased that the originality, sophistication, and scientific rationale of our research program were positively recognized. The reviewer’s assessment is highly appreciated by the authors and reinforces the relevance of integrating experimental immunomodulatory analysis with external single-cell transcriptomic contextualization to investigate intestinal inflammation during Trypanosoma cruzi infection.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript title: „ArtinM Modulates Intestinal Inflammation in Acute Experi-2 mental Trypanosoma cruzi Infection with External Single-Cell 3 Transcriptomic Contextualization” it is well written and sounds scientific.

This is a highly ambitious and scientifically mature manuscript. The author demonstrates strong expertise in immunology, molecular biology methodologies, and scRNA-seq analyses. The study has clear publication potential as a translational work integrating an in vivo experimental model with the analysis of public transcriptomic datasets.

The statistical methodology is sound, and the interpretation of the scRNA-seq data is notably mature, balanced, and appropriately cautious. Nevertheless, the manuscript requires substantial editorial revision.

The Introduction and Methods sections are excessively long and repeatedly present the same conceptual framework. Expressions such as “biologically coherent inflammatory architecture,” “independent transcriptomic context,” “without implying temporal equivalence,” and “broader inflammatory program” recur throughout the manuscript in nearly identical language. Consequently, the narrative becomes unnecessarily repetitive and overly dense.

The Introduction should be shortened by approximately 35–45%. In its current form, it reads more like a mini-review than a focused introduction to a research article.The sections most in need of condensation are the detailed description of ArtinM signaling mechanisms, the extensive immunological background, and the repeated emphasis on the “biological framework.” In particular, the information of TLR2, CD14, mast cells, neutrophils, and the various pathogen models should be condensed into a single concise paragraph.The Discussion section demonstrates a highly mature immunological interpretation of the findings and is supported by: a strong biological rationale, careful and balanced interpretation, robust integration of experimental and transcriptomic data and appropriate acknowledgment of study limitations. One of the main editorial recommendations is to substantially shorten the Discussion section, ideally by approximately 35–40%. The current version contains numerous repetitions of the same interpretative concepts, which weakens the clarity and flow of the narrative. In particular, repeated expressions such as “biologically coherent,” “transcriptomic contextualization,” “not temporally matched,” and “broader inflammatory program” should be minimized, as these ideas are already sufficiently established in earlier parts of the discussion. A more concise and focused discussion would significantly improve readability, strengthen the impact of the findings, and give the manuscript a more polished.

Author Response

Reviewer 2

 

Reviewer comment:

Manuscript title: “ArtinM Modulates Intestinal Inflammation in Acute Experimental Trypanosoma cruzi Infection with External Single-Cell Transcriptomic Contextualization” is well written and sounds scientific. This is a highly ambitious and scientifically mature manuscript. The author demonstrates strong expertise in immunology, molecular biology methodologies, and scRNA-seq analyses. The study has clear publication potential as a translational work integrating an in vivo experimental model with the analysis of public transcriptomic datasets. The statistical methodology is sound, and the interpretation of the scRNA-seq data is notably mature, balanced, and appropriately cautious. Nevertheless, the manuscript requires substantial editorial revision.

Response: We sincerely thank Reviewer 2 for the careful, constructive, and highly encouraging evaluation of our manuscript. We are grateful for the recognition of the scientific maturity, translational potential, statistical rigor, and cautious interpretation of the scRNA-seq component of the study. We also appreciate the reviewer’s clear editorial guidance regarding the need to improve conciseness, readability, and narrative flow. In response to these comments, we performed a substantial editorial revision of the manuscript, with particular attention to reducing redundancy, shortening overly dense sections, and avoiding repeated conceptual formulations. Specifically, we revised the Introduction, Methods, and Discussion to make the text more focused, direct, and suitable for a research article, while preserving the biological rationale, methodological transparency, and balanced interpretation emphasized by the reviewer.

 

Reviewer comment:

The Introduction and Methods sections are excessively long and repeatedly present the same conceptual framework. Expressions such as “biologically coherent inflammatory architecture,” “independent transcriptomic context,” “without implying temporal equivalence,” and “broader inflammatory program” recur throughout the manuscript in nearly identical language. Consequently, the narrative becomes unnecessarily repetitive and overly dense.

Response: We thank the reviewer for this precise and constructive observation. We agree that the Introduction and Methods contained repeated formulations of the same conceptual framework, particularly regarding the role of the public scRNA-seq dataset and its relationship to the acute in vivo model. In response, we revised these sections to reduce redundancy and improve readability. Specifically, we condensed the final paragraphs of the Introduction, removed repeated expressions such as “biologically coherent inflammatory architecture,” “independent transcriptomic context,” and “broader inflammatory program,” and retained only one concise statement clarifying that the public dataset was used as supportive biological context rather than as a phase-matched comparator. In the Methods, we revised the dataset-selection, inflammatory-module, module-scoring, visualization, analytical-scope, reproducibility, and murine-model subsections to avoid restating the same rationale in different forms. These changes make the manuscript more direct and less dense while preserving the methodological caution required for interpreting the scRNA-seq component.

 

Reviewer comment:

The Introduction should be shortened by approximately 35–45%. In its current form, it reads more like a mini-review than a focused introduction to a research article. The sections most in need of condensation are the detailed description of ArtinM signaling mechanisms, the extensive immunological background, and the repeated emphasis on the “biological framework.” In particular, the information of TLR2, CD14, mast cells, neutrophils, and the various pathogen models should be condensed into a single concise paragraph.

Response: We thank the reviewer for this precise and helpful recommendation. We agree that the original Introduction was overly extensive and, in some parts, resembled a mini-review rather than a focused introduction to a research article. In response, we substantially shortened the Introduction while preserving the central scientific rationale and the order of the cited literature. Specifically, we condensed the epidemiological and gastrointestinal background, reduced the repeated emphasis on the interpretive biological framework, and merged the detailed discussion of ArtinM signaling mechanisms into a single concise paragraph. This revised paragraph now summarizes the information on TLR2, CD14, T-cell activation, neutrophil activity, mast-cell responses, dose-dependent safety, and previous pathogen models in a more focused manner. We also simplified the final rationale and objective paragraphs to make the Introduction more direct and aligned with the study aims. These revisions reduced redundancy and improved readability while retaining the key biological justification for investigating ArtinM-mediated modulation of intestinal inflammation during acute T. cruzi infection.

 

Reviewer comment:

The Discussion section demonstrates a highly mature immunological interpretation of the findings and is supported by: a strong biological rationale, careful and balanced interpretation, robust integration of experimental and transcriptomic data and appropriate acknowledgment of study limitations. One of the main editorial recommendations is to substantially shorten the Discussion section, ideally by approximately 35–40%. The current version contains numerous repetitions of the same interpretative concepts, which weakens the clarity and flow of the narrative. In particular, repeated expressions such as “biologically coherent,” “transcriptomic contextualization,” “not temporally matched,” and “broader inflammatory program” should be minimized, as these ideas are already sufficiently established in earlier parts of the discussion. A more concise and focused discussion would significantly improve readability, strengthen the impact of the findings, and give the manuscript a more polished.

Response: We sincerely thank the reviewer for this thoughtful and constructive assessment of the Discussion. We are grateful for the positive recognition of the immunological interpretation, biological rationale, integration of experimental and transcriptomic data, and balanced acknowledgment of limitations. We agree that the original version was overly long and repeated several interpretative concepts that had already been sufficiently established. In response, we substantially shortened and reorganized the Discussion to improve clarity, flow, and readability. Specifically, we reduced repeated expressions such as “biologically coherent,” “transcriptomic contextualization,” “not temporally matched,” and “broader inflammatory program”; merged overlapping paragraphs on the acute-phase rationale, ArtinM-mediated immunomodulation, scRNA-seq interpretation, and study limitations; and removed redundant statements that weakened the narrative impact. The revised Discussion preserves the main biological interpretation and the key supporting citations while presenting the findings in a more concise, focused, and polished manner.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses an interesting and relevant topic related to intestinal immunomodulation during acute experimental Trypanosoma cruzi infection. The integration of experimental immunology with transcriptomic contextualization is conceptually attractive, and the in vivo findings showing attenuation of TNF-α, IFN-γ, IL-12, and inflammatory infiltrate after ArtinM treatment are potentially important.

The manuscript is generally well written, methodologically detailed, and statistically sound. The introduction is comprehensive and demonstrates strong familiarity with ArtinM biology and Chagas disease immunopathology. In addition, the experimental procedures, blinding strategy, and statistical analyses are adequately described.

However, several important limitations should be addressed.

MAJOR REVISIONS 

1. Conceptual integration between the acute experimental model and chronic transcriptomic dataset

One of the major issues requiring correction is the conceptual integration between the experimental model and the public transcriptomic datasets used for contextual support. The in vivo model evaluates acute Trypanosoma cruzi infection, whereas the scRNA-seq datasets appear to be associated with chronic intestinal inflammation. Although this discrepancy is partially acknowledged, several sections of the manuscript still imply a stronger biological correspondence than can actually be supported. The transcriptomic analysis should be clearly presented as a contextual and exploratory framework rather than a mechanistic or temporal validation of the experimental findings. Statements suggesting a “coherent inflammatory axis” or “biological support” should be moderated to avoid overinterpretation.

2. Lack of intestinal parasite burden quantification

A major experimental limitation is the absence of direct measurements of intestinal parasite burden. The study does not include qPCR, amastigote quantification, or histological assessment of tissue parasitism. Consequently, it remains unclear whether the observed effects of ArtinM are truly related to immunomodulation or could instead reflect indirect reductions in parasite load. Ideally, parasite quantification should be incorporated into the study. If this is not feasible, the authors should explicitly acknowledge this limitation and avoid making overly strong mechanistic conclusions regarding ArtinM-mediated immune modulation.

3. Limitations associated with the small sample size

The sample size used in the study represents another important limitation. The use of only five animals per group is relatively small considering the complexity of the statistical analyses performed, particularly the MANCOVA and multivariate cytokine analyses. Although the authors justify the sample size based on previous studies, there remains a substantial risk of overfitting and reduced statistical power. This limitation should be explicitly acknowledged, and conclusions derived from multivariate analyses should be presented with greater caution.

4. Excessive manuscript length and redundancy

The manuscript is considerably longer than necessary to support the presented findings. Several sections, particularly the Introduction, transcriptomic Methods, and portions of the Results, contain repetitive explanations and overly detailed conceptual descriptions. Key ideas related to transcriptomic contextualization and inflammatory relevance are reiterated multiple times throughout the text. A reduction of approximately 25–35% of the manuscript length is recommended. A more concise writing style would substantially improve readability, clarity, and editorial impact.

5. Lack of direct mechanistic validation of ArtinM

Although the study demonstrates clear associations between ArtinM treatment and reduced inflammatory parameters, no experiments directly address the underlying immunological mechanisms. Potential receptors such as TLR2 or CD14, intracellular signaling pathways such as NF-κB, and immune-cell population analyses were not evaluated. Therefore, mechanistic conclusions should be moderated. The manuscript should focus on associations compatible with immunomodulatory activity rather than presenting definitive mechanistic interpretations that are not experimentally demonstrated.

6. Limited histopathological characterization

The histopathological assessment focuses primarily on inflammatory infiltrate and does not evaluate additional pathological features relevant to digestive Chagas disease. Important parameters such as neuronal alterations, muscular damage, fibrosis, edema, necrosis, or structural tissue remodeling were not assessed. Given the importance of neuromuscular alterations in digestive Chagas pathology, this limits the biological depth of the histological interpretation. Expanding the histopathological analysis or explicitly acknowledging this limitation would strengthen the manuscript.

7. Overinterpretation of IL-10 findings

The interpretation of IL-10 results requires greater caution. IL-10 did not reach statistical significance in the conventional ANOVA analysis but became significant after adjustment using MANCOVA. Considering the small sample size and the complexity of the statistical model, this result may reflect statistical sensitivity rather than a robust biological effect. The authors should therefore present these findings more cautiously and avoid definitive conclusions regarding anti-inflammatory regulation mediated by IL-10.

 

MINOR REVISIONS 

1. Reduction and simplification of the Introduction

The Introduction contains relevant and well-supported information; however, several sections could be simplified to improve manuscript flow. The discussions regarding Chagas disease pathophysiology, ArtinM immunobiology, and transcriptomic rationale contain some conceptual redundancy. A more concise introduction would improve readability and help readers identify the study objectives more clearly.

2. Improve fluency and readability of scientific English

Although the scientific English is generally appropriate, the writing style is frequently overly dense and excessively formal. Several sentences are unnecessarily long and structurally complex, making continuous reading more difficult. Simplifying sentence structure and reducing excessive formality would improve readability without compromising scientific rigor, particularly in the Discussion and transcriptomic contextualization sections.

3. Simplification of the bioinformatics section

The transcriptomic methods and results could be simplified and summarized more effectively. At present, the level of methodological detail and descriptive emphasis conveys greater analytical complexity than what was actually performed, considering that the transcriptomic component is mainly exploratory and contextual. A more direct presentation would help maintain the primary focus on the experimental findings.

4. Explicit inclusion of study limitations

A dedicated subsection addressing study limitations is strongly recommended. Although some limitations are indirectly mentioned, they should be explicitly acknowledged in a structured manner. These include the temporal mismatch between the acute model and chronic datasets, the absence of parasite burden quantification, the small sample size, and the lack of mechanistic validation. This addition would improve methodological transparency.

5. Optimization of figures

The figures contain valuable information, although some could be visually simplified to facilitate interpretation. In particular, the transcriptomic figures would benefit from a more compact and schematic presentation. Improvements in graphical resolution and visual consistency would also enhance the overall editorial quality of the manuscript.

6. Clarification of the study novelty

The scientific novelty of the study should be highlighted more clearly. At present, the manuscript mainly emphasizes the reduction of inflammation by ArtinM; however, greater emphasis could be placed on the intestinal context, the experimental-transcriptomic integration, and the potential immunomodulatory implications of ArtinM in digestive Chagas disease.

7. Terminological consistency

Multiple similar terms are used throughout the manuscript to describe intestinal inflammation and inflammatory activation, which may generate conceptual redundancy. Standardizing terminology would improve narrative consistency and scientific readability.

Author Response

Reviewer 3

Reviewer 3 comment:

The manuscript addresses an interesting and relevant topic related to intestinal immunomodulation during acute experimental Trypanosoma cruzi infection. The integration of experimental immunology with transcriptomic contextualization is conceptually attractive, and the in vivo findings showing attenuation of TNF-α, IFN-γ, IL-12, and inflammatory infiltrate after ArtinM treatment are potentially important.
The manuscript is generally well written, methodologically detailed, and statistically sound. The introduction is comprehensive and demonstrates strong familiarity with ArtinM biology and Chagas disease immunopathology. In addition, the experimental procedures, blinding strategy, and statistical analyses are adequately described.
However, several important limitations should be addressed before publication.

Response: We sincerely thank Reviewer 3 for the careful, constructive, and thoughtful evaluation of our manuscript. We are grateful for the positive recognition of the relevance of the topic, the conceptual value of integrating experimental immunology with transcriptomic contextualization, and the potential importance of the in vivo findings showing attenuation of TNF-α, IFN-γ, IL-12, and inflammatory infiltrate after ArtinM treatment. We also appreciate the reviewer’s acknowledgment that the manuscript is generally well written, methodologically detailed, statistically sound, and adequately describes the experimental procedures, blinding strategy, and statistical analyses.

We fully agree that several limitations required clearer and more explicit treatment before publication. In response to the reviewer’s comments, we revised the manuscript to improve conceptual precision, moderate mechanistic interpretations, clarify the exploratory role of the transcriptomic component, explicitly acknowledge experimental and analytical limitations, and reduce redundancy throughout the text. We believe these revisions have substantially strengthened the clarity, balance, and methodological transparency of the manuscript.

 

Reviewer comment:

Conceptual integration between the acute experimental model and chronic transcriptomic dataset. One of the major issues requiring correction is the conceptual integration between the experimental model and the public transcriptomic datasets used for contextual support. The in vivo model evaluates acute Trypanosoma cruzi infection, whereas the scRNA-seq datasets appear to be associated with chronic intestinal inflammation. Although this discrepancy is partially acknowledged, several sections of the manuscript still imply a stronger biological correspondence than can actually be supported. The transcriptomic analysis should be clearly presented as a contextual and exploratory framework rather than a mechanistic or temporal validation of the experimental findings. Statements suggesting a “coherent inflammatory axis” or “biological support” should be moderated to avoid overinterpretation.

Response: We thank the reviewer for this important and precise observation. We agree that the previous version of the manuscript, although acknowledging the difference between the acute in vivo model and the chronic public scRNA-seq dataset, still contained some formulations that could imply a stronger biological, temporal, or mechanistic correspondence than was supported by the study design. In response, we revised the manuscript throughout to clearly define the transcriptomic component as an exploratory and contextual analysis rather than as a validation of the acute experimental findings.

Specifically, we revised the Abstract, Introduction, Methods, Results, Discussion, and Conclusions to moderate statements suggesting a “coherent inflammatory axis,” “biological support,” or direct correspondence between the two datasets. We now explicitly state that the public scRNA-seq dataset was derived from chronic infection and was not used as a phase-matched comparator, mechanistic validation, or temporal extension of the acute model. The transcriptomic analysis is now described as providing exploratory pathway-level context by assessing whether inflammatory, innate immune, chemotactic, and adhesion-associated genes related to the in vivo readouts were detectable in intestinal single-cell data from T. cruzi infection. We also revised the Results and Discussion to avoid overinterpretation and to emphasize that these transcriptomic findings do not establish causality or temporal equivalence. These changes were made to preserve the value of the integrative approach while clearly delimiting its exploratory and contextual scope.

Reviewer comment:

Lack of intestinal parasite burden quantification. A major experimental limitation is the absence of direct measurements of intestinal parasite burden. The study does not include qPCR, amastigote quantification, or histological assessment of tissue parasitism. Consequently, it remains unclear whether the observed effects of ArtinM are truly related to immunomodulation or could instead reflect indirect reductions in parasite load. Ideally, parasite quantification should be incorporated into the study. If this is not feasible, the authors should explicitly acknowledge this limitation and avoid making overly strong mechanistic conclusions regarding ArtinM-mediated immune modulation.

Response: We thank the reviewer for this important observation. We agree that the absence of direct intestinal parasite burden quantification is a relevant limitation of the present study. In the current experimental design, intestinal parasitism was not assessed by qPCR, amastigote quantification, immunohistochemistry, or histological evaluation of tissue parasitism. Therefore, we cannot determine whether the attenuation of intestinal cytokines and inflammatory infiltrate observed after ArtinM treatment resulted exclusively from host immunomodulation or was also influenced by possible reductions in local parasite load.

We note that previous work using a related acute T. cruzi infection model showed that ArtinM administration reduced peripheral parasitemia and decreased amastigote nests and inflammatory infiltrates in cardiac tissue. This prior evidence provides biological rationale for investigating ArtinM in acute Chagas disease, but we fully acknowledge that it does not replace direct assessment of parasite burden in the intestinal compartment in the present study. Accordingly, we revised the manuscript to explicitly state this limitation and to avoid overly strong mechanistic conclusions regarding ArtinM-mediated intestinal immunomodulation.

Specifically, we modified the Results, Discussion, Limitations, and Conclusions to clarify that the present findings should be interpreted as evidence of attenuation of intestinal inflammatory activity associated with ArtinM treatment, rather than as proof of direct local antiparasitic activity or definitive mechanistic immunomodulation in intestinal tissue. We also added that future studies should directly assess intestinal parasite burden using qPCR, amastigote quantification, immunohistochemistry, or related approaches to determine whether ArtinM-associated intestinal effects are driven primarily by host immune modulation, reduced tissue parasitism, or both.

 

Reviewer comment:

Limitations associated with the small sample size. The sample size used in the study represents another important limitation. The use of only five animals per group is relatively small considering the complexity of the statistical analyses performed, particularly the MANCOVA and multivariate cytokine analyses. Although the authors justify the sample size based on previous studies, there remains a substantial risk of overfitting and reduced statistical power. This limitation should be explicitly acknowledged, and conclusions derived from multivariate analyses should be presented with greater caution.

Response: We thank the reviewer for this important methodological observation. We agree that the number of animals per group is limited, particularly for adjusted multivariate analyses. At the same time, we would like to clarify that the primary conclusions of the study were not based solely on the MANCOVA. The main findings were derived from predefined tissue and cytokine endpoints, including intestinal inflammatory infiltrate and tissue concentrations of TNF-α, IFN-γ, IL-12, and IL-10, analyzed using distribution-appropriate univariate comparisons. These primary analyses showed marked group differences for TNF-α, IFN-γ, IL-12, and inflammatory infiltrate.

The MANCOVA was included as a complementary adjusted analysis to examine whether the cytokine profile remained associated with experimental group after accounting for inflammatory infiltrate. We agree that, given the sample size, this multivariate analysis should not be overinterpreted as definitive mechanistic evidence. Accordingly, we revised the manuscript to make the interpretive hierarchy clearer: the univariate inflammatory and histological outcomes are presented as the primary basis of the findings, whereas the MANCOVA is described as a complementary adjusted analysis.

In response to the reviewer’s comment, we also revised the Results and Discussion to moderate statements derived from the multivariate analysis. Expressions such as “strong evidence,” “robust separation,” and “intrinsically altered” were removed or replaced with more measured wording. In addition, we explicitly added the limited sample size to the limitations section, noting that it reduces the precision of adjusted multivariate estimates and that the MANCOVA should be interpreted as supportive of the primary findings rather than as the sole basis for mechanistic inference.

 

Reviewer comment:

Excessive manuscript length and redundancy. The manuscript is considerably longer than necessary to support the presented findings. Several sections, particularly the Introduction, transcriptomic Methods, and portions of the Results, contain repetitive explanations and overly detailed conceptual descriptions. Key ideas related to transcriptomic contextualization and inflammatory relevance are reiterated multiple times throughout the text. A reduction of approximately 25–35% of the manuscript length is recommended. A more concise writing style would substantially improve readability, clarity, and editorial impact.

Response: We thank the reviewer for this important editorial recommendation. We agree that the previous version of the manuscript contained excessive length and conceptual redundancy, particularly in the Introduction, transcriptomic Methods, Results, and Discussion. In response, we performed a substantial editorial revision throughout the manuscript to improve conciseness, readability, and narrative flow.

Specifically, we shortened the Introduction by condensing the background on Chagas disease, ArtinM immunobiology, and the rationale for using public transcriptomic data. We also simplified the transcriptomic Methods by reducing repeated explanations regarding the public scRNA-seq dataset, its exploratory purpose, and its lack of temporal correspondence with the acute experimental model. In the Results, we revised the transcriptomic subsections to present the analyses more directly, reduced overly detailed descriptive passages, and moderated interpretive language related to inflammatory relevance and transcriptomic contextualization. The Discussion was also reorganized and shortened to minimize repeated interpretative concepts and improve the overall flow of the manuscript.

In addition, we standardized terminology throughout the text and reduced recurrent expressions such as “coherent inflammatory axis,” “biological support,” “broader inflammatory program,” and “transcriptomic contextualization.” The revised manuscript now presents the in vivo experimental findings as the primary contribution, while the public scRNA-seq analysis is clearly framed as exploratory pathway-level context. We believe these changes substantially improve clarity, readability, and editorial focus while preserving the scientific rationale, methodological transparency, and main findings of the study.

 

Reviewer comment:

Lack of direct mechanistic validation of ArtinM. Although the study demonstrates clear associations between ArtinM treatment and reduced inflammatory parameters, no experiments directly address the underlying immunological mechanisms. Potential receptors such as TLR2 or CD14, intracellular signaling pathways such as NF-κB, and immune-cell population analyses were not evaluated. Therefore, mechanistic conclusions should be moderated. The manuscript should focus on associations compatible with immunomodulatory activity rather than presenting definitive mechanistic interpretations that are not experimentally demonstrated.

Response: We thank the reviewer for this important and appropriate comment. We agree that the present study does not directly test the receptor-level, signaling-level, or cellular mechanisms underlying the intestinal effects associated with ArtinM treatment. Specifically, we did not evaluate TLR2, CD14, MyD88, NF-κB activation, or immune-cell population dynamics in intestinal tissue. Therefore, we agree that mechanistic interpretations should be framed cautiously.

In response, we revised the manuscript to moderate mechanistic language throughout the Results, Discussion, Limitations, and Conclusions. We now describe the observed reductions in TNF-α, IFN-γ, IL-12, and inflammatory infiltrate as inflammatory changes associated with ArtinM treatment and compatible with immunomodulatory activity, rather than as direct proof of a specific mechanistic pathway. We also clarified that references to TLR2, CD14, MyD88, NF-κB, PI3K, and T-cell-related pathways are based on previous ArtinM literature and are used only to provide biological context and generate hypotheses for future mechanistic studies.

We added an explicit limitation stating that receptor-level mechanisms, intracellular signaling pathways, and immune-cell population analyses were not directly evaluated in the present intestinal model. Accordingly, the revised manuscript avoids definitive mechanistic conclusions and emphasizes that future studies should directly assess local parasite burden, receptor- and pathway-level activation, and immune-cell population dynamics to determine the mechanisms underlying ArtinM-associated attenuation of intestinal inflammation.

 

Reviewer comment:

Limited histopathological characterization. The histopathological assessment focuses primarily on inflammatory infiltrate and does not evaluate additional pathological features relevant to digestive Chagas disease. Important parameters such as neuronal alterations, muscular damage, fibrosis, edema, necrosis, or structural tissue remodeling were not assessed. Given the importance of neuromuscular alterations in digestive Chagas pathology, this limits the biological depth of the histological interpretation. Expanding the histopathological analysis or explicitly acknowledging this limitation would strengthen the manuscript.

Response: We thank the reviewer for this important observation. We agree that the histopathological assessment in the present study was focused on morphometric quantification of intestinal inflammatory infiltrate and did not include a broader evaluation of digestive Chagas disease-related tissue pathology, such as neuronal alterations, muscular damage, fibrosis, edema, necrosis, structural remodeling, or neuromuscular preservation.

We would like to clarify that the present study was designed as an acute experimental model focused primarily on intestinal inflammatory outcomes. Accordingly, inflammatory infiltrate was defined as the primary tissue-level histomorphometric endpoint, while intestinal concentrations of TNF-α, IFN-γ, IL-12, and IL-10 were used as tissue inflammatory readouts. Comprehensive assessment of neuromuscular alterations and structural remodeling was beyond the scope of the current acute-phase design and would be more appropriately addressed in future studies focused on digestive tissue progression or chronic outcomes.

In response to the reviewer’s comment, we revised the manuscript to make this scope explicit. We clarified in the Methods that the histopathological assessment was restricted to inflammatory infiltrate quantification. We also revised the Discussion to avoid overinterpreting the findings as evidence of comprehensive digestive tissue protection or neuromuscular preservation. In addition, we added this limitation to the limitations section and revised the Conclusions to indicate that future studies should include expanded histopathological characterization, enteric neuronal assessment, structural remodeling analysis, and chronic digestive outcomes. These revisions preserve the inflammatory focus of the present acute model while acknowledging the need for broader histopathological characterization in future studies.

 

Reviewer comment:

Overinterpretation of IL-10 findings. The interpretation of IL-10 results requires greater caution. IL-10 did not reach statistical significance in the conventional ANOVA analysis but became significant after adjustment using MANCOVA. Considering the small sample size and the complexity of the statistical model, this result may reflect statistical sensitivity rather than a robust biological effect. The authors should therefore present these findings more cautiously and avoid definitive conclusions regarding anti-inflammatory regulation mediated by IL-10.

Response: We thank the reviewer for this important and precise observation. We agree that the IL-10 finding requires cautious interpretation. In the present study, IL-10 did not reach statistical significance in the primary univariate ANOVA and became significant only in the adjusted MANCOVA model, in which inflammatory infiltrate was included as a covariate. We would like to clarify that the use of MANCOVA was biologically and statistically motivated, since inflammatory infiltrate represents local tissue inflammatory burden and may reasonably influence cytokine concentrations in intestinal tissue. Therefore, inflammatory infiltrate was included as a covariate to examine whether the cytokine profile remained associated with experimental group after accounting for this histological component.

At the same time, we agree that the adjusted IL-10 result should not be interpreted as definitive evidence of IL-10-mediated anti-inflammatory regulation, particularly given the limited sample size and the complementary nature of the adjusted multivariate analysis. In response, we revised the Results to clarify that the adjusted IL-10 finding suggests a possible association between IL-10 variation, experimental group, and inflammatory infiltrate after statistical adjustment, but should be considered hypothesis-generating rather than conclusive. We also revised the Discussion to emphasize that the main measurable effect associated with ArtinM treatment was the attenuation of TNF-α, IFN-γ, IL-12, and inflammatory infiltrate, whereas IL-10 should be interpreted as a secondary adjusted signal.

In addition, we added a specific statement to the limitations section noting that IL-10 did not reach significance in the primary univariate analysis and should not be taken as conclusive evidence of IL-10-mediated regulation. These revisions preserve the biological and statistical rationale for adjusting cytokine outcomes by inflammatory infiltrate while aligning the interpretation of IL-10 with the strength of the evidence generated by the study.

.

Minor revision

Reviewer comment:

Reduction and simplification of the Introduction. The Introduction contains relevant and well-supported information; however, several sections could be simplified to improve manuscript flow. The discussions regarding Chagas disease pathophysiology, ArtinM immunobiology, and transcriptomic rationale contain some conceptual redundancy. A more concise introduction would improve readability and help readers identify the study objectives more clearly.

Response: We thank the reviewer for this helpful recommendation. We revised the Introduction to improve conciseness, flow, and clarity. Specifically, we condensed the background on Chagas disease pathophysiology, simplified the discussion of ArtinM immunobiology, and reduced repeated statements related to the transcriptomic rationale. The revised Introduction now presents the intestinal focus, the acute experimental model, and the study objectives more directly, while preserving the essential biological rationale and supporting references.

 

Reviewer comment:

Improve fluency and readability of scientific English. Although the scientific English is generally appropriate, the writing style is frequently overly dense and excessively formal. Several sentences are unnecessarily long and structurally complex, making continuous reading more difficult. Simplifying sentence structure and reducing excessive formality would improve readability without compromising scientific rigor, particularly in the Discussion and transcriptomic contextualization sections.

Response: We thank the reviewer for this important editorial suggestion. We revised the manuscript to improve fluency, readability, and sentence structure, with particular attention to the Discussion and transcriptomic sections. Long and overly dense sentences were simplified, excessive formality was reduced, and repeated interpretive formulations were removed or shortened. These changes were made to improve readability while maintaining scientific precision and methodological rigor.

 

Reviewer comment:

Simplification of the bioinformatics section. The transcriptomic methods and results could be simplified and summarized more effectively. At present, the level of methodological detail and descriptive emphasis conveys greater analytical complexity than what was actually performed, considering that the transcriptomic component is mainly exploratory and contextual. A more direct presentation would help maintain the primary focus on the experimental findings.

Response: We agree with the reviewer and revised the bioinformatics-related sections accordingly. The transcriptomic Methods and Results were simplified to emphasize that the public scRNA-seq analysis was exploratory and contextual rather than mechanistic or confirmatory. We reduced excessive methodological detail, moderated interpretive language, and presented the gene-module and inflammatory-score analyses more directly. The revised text now maintains the primary focus on the in vivo experimental findings while retaining sufficient information for reproducibility and transparency.

 

Reviewer comment:

Explicit inclusion of study limitations. A dedicated subsection addressing study limitations is strongly recommended. Although some limitations are indirectly mentioned, they should be explicitly acknowledged in a structured manner. These include the temporal mismatch between the acute model and chronic datasets, the absence of parasite burden quantification, the small sample size, and the lack of mechanistic validation. This addition would improve methodological transparency.

Response: We thank the reviewer for this recommendation. We added an explicit and structured limitations paragraph in the Discussion. This section now directly addresses the absence of intestinal parasite burden quantification, the limited number of animals per group, the complementary nature of the MANCOVA, the cautious interpretation of IL-10, the lack of direct receptor-level and pathway-level mechanistic validation, the restricted histopathological scope, the temporal mismatch between the acute in vivo model and chronic public scRNA-seq dataset, and the lack of reliable lineage-resolved cell-type annotation in the processed public metadata. This revision improves methodological transparency and clearly defines the interpretive scope of the study.

Reviewer comment:

Optimization of figures. The figures contain valuable information, although some could be visually simplified to facilitate interpretation. In particular, the transcriptomic figures would benefit from a more compact and schematic presentation. Improvements in graphical resolution and visual consistency would also enhance the overall editorial quality of the manuscript.

Response: We thank the reviewer for this helpful suggestion. We revised the figures and figure descriptions to improve clarity, consistency, and interpretability. In particular, the transcriptomic figures were maintained as compact summaries to reinforce their exploratory contextual role and to avoid overemphasizing the bioinformatics component relative to the experimental findings. We also reviewed graphical resolution, labeling, and visual consistency across the figures to improve the overall editorial quality of the manuscript.

 

Reviewer comment: Clarification of the study novelty. The scientific novelty of the study should be highlighted more clearly. At present, the manuscript mainly emphasizes the reduction of inflammation by ArtinM; however, greater emphasis could be placed on the intestinal context, the experimental-transcriptomic integration, and the potential immunomodulatory implications of ArtinM in digestive Chagas disease.

Response: We agree with the reviewer and revised the manuscript to clarify the novelty of the study. The revised text now emphasizes that the main contribution is not only the attenuation of inflammatory parameters by ArtinM, but also the direct assessment of intestinal inflammatory outcomes during acute experimental T. cruzi infection. We also clarified that the public scRNA-seq analysis was used as exploratory pathway-level context for related inflammatory pathways. Together, these revisions better highlight the intestinal focus, the experimental-transcriptomic integration, and the potential immunomodulatory implications of ArtinM in the context of digestive Chagas disease.

 

Reviewer comment: Terminological consistency. Multiple similar terms are used throughout the manuscript to describe intestinal inflammation and inflammatory activation, which may generate conceptual redundancy. Standardizing terminology would improve narrative consistency and scientific readability.

Response: We thank the reviewer for this observation. We standardized terminology throughout the manuscript to improve narrative consistency and reduce conceptual redundancy. Recurrent expressions such as “coherent inflammatory axis,” “biological support,” “broader inflammatory program,” and “transcriptomic contextualization” were removed or replaced with more precise terminology. We now consistently use terms such as “intestinal inflammatory response,” “inflammatory infiltrate,” “inflammatory outcomes,” “public scRNA-seq analysis,” and “exploratory pathway-level context.” These changes improved readability and aligned the terminology with the revised interpretive scope of the study.

 

Reviewer 4 Report

Comments and Suggestions for Authors

This study evaluated whether ArtinM modulates intestinal inflammatory responses during acute Trypanosoma cruzi infection and interpreted these effects within an external intestinal single-cell transcriptomic context. The study is well-conceived, methodologically robust, and provides valuable insights into the immunomodulatory role of ArtinM and the organization of intestinal inflammatory responses during infection. The work is clearly written, scientifically sound, and represents a meaningful contribution to the understanding of host–pathogen interactions in Chagas disease, requiring only minor refinements.

 

Special attention should be given to the correct formatting of species names according to standard scientific nomenclature conventions. The same applies to the expression in vivo.

 

Overall, the manuscript is somewhat repetitive, with instances of redundancy. I recommend reducing textual repetition and simplifying some overly long sentences to improve clarity and readability.

 

Please clarify which form of IL-12 was measured. IL-12p70 or total IL-12?

 

Introduction

• There is some conceptual repetition, particularly regarding the importance of the acute phase, the distinction between the quality and magnitude of inflammation, and the value of transcriptomic analysis. These aspects should be streamlined.
• Although the objectives are clearly stated, the study hypothesis is not explicitly formulated and should be clearly defined.

 

Materials and Methods

• It is not entirely clear whether raw or normalized counts were used in the transcriptomic analysis; this should be explicitly specified.
• In subsection 2.5 (Single-cell inflammatory module scoring), the nature of the score (InflammatoryScore1) should be briefly explained (represents a mean? sum?).
• The unit “pg/mL/g tissue” appears inconsistent with the statement that values were normalized to tissue mass. This should be revised to a more appropriate unit (e.g., pg/g tissue).
• For consistency and readability, I suggest the following terminology adjustments:

“Saline” → “Saline control”

“Saline + T. cruzi” → “T. cruzi + Saline”

“Saline + T. cruzi > ArtinM + T. cruzi” → “Saline + T. cruzi vs. ArtinM + T. cruzi”

 

Tables

• In Table 1, inflammatory infiltrate (%) is reported as mean ± SD, although it was analyzed using a non-parametric test (Kruskal–Wallis). This is inconsistent.
• In Table 2, for consistency with the text, the authors should include Hotelling’s and Roy’s in the table.

Author Response

Reviewer 4

 

Reviewer comment:

This study evaluated whether ArtinM modulates intestinal inflammatory responses during acute Trypanosoma cruzi infection and interpreted these effects within an external intestinal single-cell transcriptomic context. The study is well-conceived, methodologically robust, and provides valuable insights into the immunomodulatory role of ArtinM and the organization of intestinal inflammatory responses during infection. The work is clearly written, scientifically sound, and represents a meaningful contribution to the understanding of host–pathogen interactions in Chagas disease, requiring only minor refinements.

Response: We sincerely thank Reviewer 4 for the thoughtful, encouraging, and constructive evaluation of our manuscript. We are grateful for the reviewer’s recognition of the study as well-conceived, methodologically robust, scientifically sound, and relevant to the understanding of host–pathogen interactions in Chagas disease. We also appreciate the positive assessment of the intestinal focus, the ArtinM-associated inflammatory findings, and the integration with public single-cell transcriptomic context. In response to the reviewer’s minor recommendations, we carefully revised the manuscript to improve nomenclature consistency, methodological clarity, terminology, table formatting, and overall readability. We believe these refinements have strengthened the revised version while preserving the central scientific contribution of the study.

 

Reviewer comment:

Special attention should be given to the correct formatting of species names according to standard scientific nomenclature conventions. The same applies to the expression in vivo.

Response: We thank the reviewer for this important editorial observation. We revised the manuscript to ensure that species names follow standard scientific nomenclature conventions. Specifically, we italicized all occurrences of Trypanosoma cruzi, T. cruzi, and other scientific names mentioned in the manuscript, including Artocarpus heterophyllus, Candida albicans, Neospora caninum, Toxoplasma gondii, and Cryptococcus gattii, where applicable. We also reviewed the manuscript for consistency in the formatting of the expression in vivo and applied italics throughout the text. In addition, we standardized the expression in silico by removing hyphenated forms and applying consistent formatting. These corrections were applied across the title, abstract, main text, tables, figure legends, and abbreviations where relevant.

 

Reviewer comment:

Overall, the manuscript is somewhat repetitive, with instances of redundancy. I recommend reducing textual repetition and simplifying some overly long sentences to improve clarity and readability.

Response: We thank the reviewer for this helpful editorial recommendation. We agree that the previous version contained some redundant formulations and overly long sentences that affected readability. In response, we performed a substantial editorial revision throughout the manuscript to reduce textual repetition, simplify sentence structure, and improve narrative flow.

Specifically, we shortened and streamlined the Introduction, reduced repeated explanations in the transcriptomic Methods and Results, and reorganized the Discussion to avoid recurring interpretive statements. We also standardized terminology related to intestinal inflammation and transcriptomic contextualization, replacing repeated expressions with more concise and consistent wording. These revisions improved clarity and readability while preserving the scientific rationale, methodological transparency, and main findings of the study.

 

Reviewer comment:

Please clarify which form of IL-12 was measured. IL-12p70 or total IL-12?

Response: We thank the reviewer for this important observation. We agree that the previous version did not clearly specify which IL-12-related analyte was measured. We revised the Methods section to clarify that the ELISA assay used in this study measured IL-12p40, not heterodimeric IL-12p70.

Accordingly, we replaced “IL-12” with “IL-12p40” throughout the manuscript wherever referring to the experimentally measured protein, including the Abstract, Methods, Results, Tables, Discussion, and Conclusions. We also revised the interpretation of this marker to avoid implying that bioactive IL-12p70 was directly measured. In the revised manuscript, IL-12p40 is interpreted as a p40-associated inflammatory signal, given that p40 is a subunit shared by IL-12 and IL-23. In the transcriptomic section, we retained the gene symbols Il12a and Il12b, while clarifying that Il12b encodes the p40 subunit and that these genes represent IL-12-related transcriptomic components rather than direct protein-level IL-12p70 measurement.

 

Reviewer comment:

Introduction. There is some conceptual repetition, particularly regarding the importance of the acute phase, the distinction between the quality and magnitude of inflammation, and the value of transcriptomic analysis. These aspects should be streamlined. Although the objectives are clearly stated, the study hypothesis is not explicitly formulated and should be clearly defined.

Response: We thank the reviewer for this helpful recommendation. We revised the Introduction to reduce conceptual repetition regarding the acute phase, the qualitative versus quantitative interpretation of inflammation, and the role of the transcriptomic analysis. In the revised version, the discussion of the acute model and the public scRNA-seq dataset was streamlined to clarify that the transcriptomic component was used only as exploratory pathway-level context, not as a temporally matched comparator or validation dataset.

We also agree that the study hypothesis should be explicitly stated. Accordingly, we added a clear hypothesis before the study objective: that ArtinM treatment would attenuate the acute intestinal inflammatory response induced by T. cruzi infection, as reflected by reduced inflammatory infiltrate and lower tissue concentrations of pro-inflammatory cytokines. This revision makes the logical transition from background rationale to hypothesis, objectives, and novelty more direct and improves the clarity of the Introduction.

 

Materials and Methods

Reviewer comment:

It is not entirely clear whether raw or normalized counts were used in the transcriptomic analysis; this should be explicitly specified.

Response: We thank the reviewer for this important methodological observation. We revised the Methods section to explicitly clarify how expression matrices were used in the transcriptomic analysis.

Gene-level detection and quantitative expression summaries were derived from count matrices extracted from the processed Seurat objects. In the analytical workflow, the RNA counts layer was prioritized when available, with the RNA data layer used only as a fallback if counts were not accessible. These count-based summaries were used to determine whether each inflammatory-module gene was present, to calculate the number and percentage of expressing cells, and to summarize mean expression values across all cells and among expressing cells only.

We also clarified that the composite inflammatory score was handled differently. The InflammatoryScore1 variable was generated after normalization of the Seurat object using NormalizeData() and module scoring with AddModuleScore(), and was then extracted from the processed metadata and analyzed as provided. These revisions make the transcriptomic workflow more transparent and clarify the descriptive and exploratory nature of the public scRNA-seq analysis.

 

Reviewer comment:

In subsection 2.5 (Single-cell inflammatory module scoring), the nature of the score (InflammatoryScore1) should be briefly explained (represents a mean? sum?).

Response: We thank the reviewer for this important clarification request. We revised subsection 2.5 to explicitly explain the nature of InflammatoryScore1. The score is not a raw count sum or a simple arithmetic mean of gene counts. It is a Seurat-derived module score generated after normalization of the Seurat object using NormalizeData() and module scoring with AddModuleScore().

In the revised Methods, we now clarify that InflammatoryScore1 represents a per-cell module score calculated from normalized expression values of the predefined inflammatory gene set relative to matched control genes. We also state that this score was extracted from the processed metadata and analyzed as provided, serving only as a descriptive per-cell summary of inflammatory-module activity in the exploratory transcriptomic component.

 

Reviewer comment:

The unit “pg/mL/g tissue” appears inconsistent with the statement that values were normalized to tissue mass. This should be revised to a more appropriate unit (e.g., pg/g tissue).

Response: We thank the reviewer for identifying this inconsistency. We agree that, because cytokine concentrations were normalized to tissue mass, the unit “pg/mL/g tissue” was not the most appropriate way to report the results. We revised the Methods, Results, Table 1, and Table 2 to express tissue-normalized cytokine concentrations as “pg/g tissue.” This correction was applied consistently to TNF-α, IFN-γ, IL-12p40, and IL-10 throughout the manuscript. The numerical values were not changed; only the unit description was revised to accurately reflect mass-normalized cytokine concentrations.

 

 

 

Reviewer comment:

For consistency and readability, I suggest the following terminology adjustments: “Saline” → “Saline control”; “Saline + T. cruzi” → “T. cruzi + Saline”; “Saline + T. cruzi > ArtinM + T. cruzi” → “Saline + T. cruzi vs. ArtinM + T. cruzi”.

Response: We thank the reviewer for this helpful suggestion. We revised the group terminology throughout the manuscript to improve consistency and readability. Specifically, “Saline” was replaced with “Saline control,” “Saline + T. cruzi” was replaced with “T. cruzi + Saline,” and “ArtinM + T. cruzi” was standardized as “T. cruzi + ArtinM.” These changes were applied in the Abstract, Methods, Results, tables, and figure/table legends where applicable.

We also revised the post hoc comparison notation in Table 1. Instead of using the “>” symbol, pairwise comparisons are now presented using “vs.” terminology, with the direction of the difference indicated in parentheses when relevant. This revision improves readability while preserving the statistical interpretation of the results.

 

Reviewer comment:

In Table 1, inflammatory infiltrate (%) is reported as mean ± SD, although it was analyzed using a non-parametric test (Kruskal–Wallis). This is inconsistent.

Response: We thank the reviewer for identifying this inconsistency. We agree that inflammatory infiltrate should be descriptively presented in a manner consistent with the non-parametric analysis used for this outcome. Because inflammatory infiltrate violated normality and homogeneity assumptions and was analyzed using the Kruskal–Wallis test followed by Dwass–Steel–Critchlow–Fligner pairwise comparisons, we revised Table 1 to report this variable as median and interquartile range rather than mean and standard deviation. The corresponding Results text was also revised to report median (IQR) values for inflammatory infiltrate. Cytokine outcomes, which were analyzed using parametric tests, remain presented as mean (SD). This revision aligns the descriptive statistics with the inferential approach used for each outcome without changing the statistical results or their interpretation.

 

Reviewer comment:

In Table 2, for consistency with the text, the authors should include Hotelling’s and Roy’s in the table.

Response: We thank the reviewer for this careful observation. We agree that Table 2 should be consistent with the multivariate tests reported in the Results section. Accordingly, we revised Table 2 to include Hotelling’s trace and Roy’s largest root, in addition to Pillai’s trace and Wilks’ lambda, for both the group effect and the inflammatory infiltrate covariate. This revision provides a complete presentation of the MANCOVA results and improves consistency between the text and the table, without changing the statistical interpretation of the findings.

 

Reviewer 5 Report

Comments and Suggestions for Authors

Overall assessment

This manuscript investigates whether ArtinM modulates intestinal inflammation during acute experimental Trypanosoma cruzi infection, with additional contextualization using external single-cell transcriptomic data. The topic is interesting and potentially relevant, particularly because gastrointestinal involvement in Chagas disease remains less explored than cardiac pathology.The in vivo data suggest that ArtinM treatment reduces intestinal TNF-α, IFN-γ, IL-12 levels and markedly decreases inflammatory infiltrates in infected mice. The external scRNA-seq analysis provides useful background showing that the cytokines measured experimentally belong to a broader intestinal inflammatory transcriptional program.However, several issues should be addressed. In particular, the manuscript should better distinguish experimental findings from external transcriptomic contextualization, avoid overinterpreting mechanisms that were not directly tested, and provide clearer methodological details for histological quantification.

Major comments
1. The manuscript shows reduced inflammatory cytokines and inflammatory infiltrates after ArtinM treatment, but it does not assess intestinal parasite burden. Therefore, it remains unclear whether the observed effects result from reduced local parasite load, direct immunomodulation, or both.

The authors should avoid implying a local anti-parasitic effect unless additional data are provided. If feasible, quantification of parasite burden by qPCR, histology, or amastigote nest counting would strengthen the study. Otherwise, the conclusions should be restricted to inflammatory modulation and partial tissue protection.

2. The discussion refers to TLR2, CD14, MyD88, NF-κB and related pathways as possible mechanisms of ArtinM action. However, these pathways were not directly measured in the present study.

The authors should revise the wording to clarify that these mechanisms are inferred from previous literature and are only compatible with, but not demonstrated by, the current data. Stronger mechanistic claims would require additional assays such as pathway-specific qPCR, immunohistochemistry, Western blotting, or protein-level signaling readouts.

3. The single-cell transcriptomic analysis uses an external chronic infection dataset, whereas the experimental model in this manuscript concerns acute infection. This distinction is important and should be made more explicit throughout the manuscript, including in the abstract, results, discussion, and conclusion.

The scRNA-seq analysis is valuable as contextualization, but it should not be presented as direct validation of the ArtinM-treated acute infection model.

4. The scRNA-seq analysis demonstrates the presence of inflammatory module genes, but without reliable cell-type annotation it remains unclear which cell populations contribute to these signals.

If possible, the authors should provide at least broad cell-type annotation using canonical markers. If this is not feasible, the limitation should be clearly stated, and the single-cell analysis should be framed as gene-module-level contextualization rather than cellular mechanism analysis.

5. The reduction in inflammatory infiltrate is one of the central findings of the manuscript. However, the histological quantification method needs further clarification.

The authors should specify the number of sections and fields analyzed per animal, whether field selection and image analysis were blinded, how inflammatory areas were defined, which software or thresholding method was used, and whether quantification was normalized to total tissue area. Representative images should be clearly linked to the quantitative results.

6. IL-10 did not reach significance in the univariate analysis but showed significance in the MANCOVA model after adjustment for inflammatory infiltrate. Given the small sample size and exploratory nature of this analysis, IL-10 should not be presented as a primary ArtinM-driven effect.

The authors should describe the IL-10 result as exploratory or hypothesis-generating and discuss it as a possible compensatory regulatory response associated with tissue inflammation.

Minor comments

The abstract should clearly state that the scRNA-seq dataset is external and derived from chronic infection, whereas the in vivo experiment uses an acute infection model.

The terminology should be standardized. Terms such as “validation,” “integration,” and “confirmation” should be avoided unless the authors can demonstrate direct experimental correspondence.

Figure legends should provide more information on sample size, statistical tests, p-value annotations, and the composition of the inflammatory gene module.

The statistical methods should more clearly describe post hoc tests, multiple-comparison correction, MANCOVA assumptions, and covariate selection.

The animal methods would benefit from additional details on randomization, blinding, humane endpoints, body weight monitoring, mortality, and clinical signs.

Author Response

Reviewer 5

 

Reviewer comment:

Overall assessment. This manuscript investigates whether ArtinM modulates intestinal inflammation during acute experimental Trypanosoma cruzi infection, with additional contextualization using external single-cell transcriptomic data. The topic is interesting and potentially relevant, particularly because gastrointestinal involvement in Chagas disease remains less explored than cardiac pathology. The in vivo data suggest that ArtinM treatment reduces intestinal TNF-α, IFN-γ, IL-12 levels and markedly decreases inflammatory infiltrates in infected mice. The external scRNA-seq analysis provides useful background showing that the cytokines measured experimentally belong to a broader intestinal inflammatory transcriptional program. However, several issues should be addressed. In particular, the manuscript should better distinguish experimental findings from external transcriptomic contextualization, avoid overinterpreting mechanisms that were not directly tested, and provide clearer methodological details for histological quantification.

Response: We sincerely thank the reviewer for the careful, constructive, and balanced assessment of our manuscript. We are grateful for the recognition of the relevance of the topic, especially regarding the intestinal component of Chagas disease, which remains less explored than cardiac pathology. We also appreciate the reviewer’s positive evaluation of the in vivo findings and of the usefulness of the external scRNA-seq dataset as contextual background.

We fully agree that the manuscript required clearer separation between direct experimental findings and external transcriptomic contextualization, more cautious wording regarding mechanisms not directly tested, and additional methodological detail for histological quantification. In response, we revised the manuscript extensively to address these points. Specifically, we clarified throughout the Abstract, Results, Discussion, and Conclusions that the scRNA-seq analysis was external, derived from a chronic infection dataset, and used only as exploratory pathway-level context rather than as direct validation of the acute in vivo model. We also moderated mechanistic language related to TLR2, CD14, MyD88, NF-κB, and related pathways, framing these mechanisms as literature-based biological plausibility rather than as mechanisms demonstrated in the present study. In addition, we expanded the methodological description of histological quantification, including the number of fields analyzed per animal, blinding procedures, morphometric approach, software used, and normalization to tissue area.

We believe these revisions substantially improved the clarity, methodological transparency, and interpretive balance of the manuscript while preserving the central experimental contribution of the study.

 

Major comments

Reviewer comment:

The manuscript shows reduced inflammatory cytokines and inflammatory infiltrates after ArtinM treatment, but it does not assess intestinal parasite burden. Therefore, it remains unclear whether the observed effects result from reduced local parasite load, direct immunomodulation, or both. The authors should avoid implying a local anti-parasitic effect unless additional data are provided. If feasible, quantification of parasite burden by qPCR, histology, or amastigote nest counting would strengthen the study. Otherwise, the conclusions should be restricted to inflammatory modulation and partial tissue protection.

Response: We thank the reviewer for this important and appropriate comment. We agree that the absence of direct intestinal parasite burden quantification limits the interpretation of the intestinal effects observed after ArtinM treatment. In the present study, we did not quantify local parasite burden by qPCR, amastigote counting, immunohistochemistry, or histological assessment of tissue parasitism. Therefore, we cannot determine whether the attenuation of intestinal cytokines and inflammatory infiltrate resulted exclusively from host inflammatory modulation, possible reductions in local parasite load, or both.

In response, we revised the manuscript to avoid implying a direct local antiparasitic effect of ArtinM in the intestinal compartment. The Results, Discussion, Limitations, Abstract, and Conclusions were revised to state that the findings should be interpreted as evidence of attenuation of intestinal inflammatory outcomes rather than as proof of local antiparasitic activity. We also explicitly added this point to the limitations section and indicated that future studies should directly assess intestinal parasite burden, amastigote nests, and tissue parasitism to clarify whether the observed intestinal effects are driven by immunomodulation, reduced local parasitism, or both.

 

Reviewer comment:

The discussion refers to TLR2, CD14, MyD88, NF-κB and related pathways as possible mechanisms of ArtinM action. However, these pathways were not directly measured in the present study. The authors should revise the wording to clarify that these mechanisms are inferred from previous literature and are only compatible with, but not demonstrated by, the current data. Stronger mechanistic claims would require additional assays such as pathway-specific qPCR, immunohistochemistry, Western blotting, or protein-level signaling readouts.

Response: We thank the reviewer for this important comment. We agree that the present study did not directly evaluate receptor-level, signaling-level, or protein-level mechanisms involving TLR2, CD14, MyD88, NF-κB, or related pathways in intestinal tissue. Therefore, these pathways cannot be presented as mechanisms demonstrated by the current data.

In response, we revised the Discussion to clarify that references to TLR2, CD14, MyD88, NF-κB, PI3K, and related signaling pathways are based on previous ArtinM literature and are used only as a literature-based framework for biological plausibility. We also revised the wording to state that the observed reductions in TNF-α, IFN-γ, IL-12p40, and inflammatory infiltrate are compatible with ArtinM-associated immunomodulatory activity, but do not constitute direct evidence of a specific receptor- or pathway-mediated mechanism.

In addition, we added this point explicitly to the limitations section, stating that receptor-level mechanisms, intracellular signaling pathways, protein-level activation, and immune-cell population dynamics were not directly evaluated in the present intestinal model. We now indicate that future studies should include pathway-specific qPCR, immunohistochemistry, Western blotting, protein-level signaling readouts, and immune-cell profiling to determine the mechanisms underlying the intestinal inflammatory changes associated with ArtinM treatment.

 

Reviewer comment:

The single-cell transcriptomic analysis uses an external chronic infection dataset, whereas the experimental model in this manuscript concerns acute infection. This distinction is important and should be made more explicit throughout the manuscript, including in the abstract, results, discussion, and conclusion. The scRNA-seq analysis is valuable as contextualization, but it should not be presented as direct validation of the ArtinM-treated acute infection model.

Response: We thank the reviewer for this important comment. We fully agree that the external scRNA-seq dataset and the acute experimental model must be clearly distinguished. In response, we revised the manuscript throughout to make this distinction explicit and to avoid presenting the transcriptomic analysis as validation of the ArtinM-treated acute infection model.

Specifically, we revised the Abstract to state that the public murine intestinal scRNA-seq dataset was external, derived from a chronic infection setting, and used only to provide exploratory pathway-level context. We also clarified that it was not interpreted as a phase-matched comparator, mechanistic validation, or temporal extension of the acute experimental findings. In the Methods, we expanded the analytical scope and interpretive framework to state that no phase-matched comparison, causal inference, mechanistic validation, or cell-type-specific interpretation was performed. In the Results and Discussion, we revised the language to describe the scRNA-seq findings as descriptive gene-module-level contextualization rather than direct support or validation of the in vivo findings. Finally, in the Conclusions, we explicitly state that the transcriptomic resource was interpreted only as exploratory pathway-level context and not as temporal or mechanistic validation of the acute experimental findings.

These revisions preserve the value of the public scRNA-seq analysis as contextual information while clearly separating it from the direct experimental evidence generated in the acute in vivo model.

 

 

Reviewer comment:

The scRNA-seq analysis demonstrates the presence of inflammatory module genes, but without reliable cell-type annotation it remains unclear which cell populations contribute to these signals. If possible, the authors should provide at least broad cell-type annotation using canonical markers. If this is not feasible, the limitation should be clearly stated, and the single-cell analysis should be framed as gene-module-level contextualization rather than cellular mechanism analysis.

Response: We thank the reviewer for this important comment. We agree that, without reliable lineage-resolved cell-type annotation, the scRNA-seq analysis cannot identify which specific cell populations contributed to the inflammatory-module signals. In the present analysis, the processed public metadata did not support robust biologically resolved cell-type annotation, and therefore we did not consider it appropriate to infer cell-specific inflammatory mechanisms from these objects.

In response, we revised the manuscript to make this limitation explicit and to frame the scRNA-seq analysis strictly as gene-module-level contextualization. Specifically, we clarified in the Methods that the public objects were interpreted as global single-cell transcriptomic maps rather than definitive cell-type annotation maps. We also revised the Results to state that the inflammatory gene-module findings describe gene presence and expression patterns across the processed objects, not inflammatory activity in defined immune-cell populations. In the Discussion and limitations section, we further emphasized that the absence of reliable lineage-resolved annotation prevents cell-type-specific interpretation.

These revisions ensure that the scRNA-seq component is presented as exploratory pathway-level and gene-module-level context, not as a cellular mechanism analysis. Future studies with curated cell-type annotation, canonical marker validation, or experimentally generated single-cell data from the acute ArtinM-treated model will be necessary to determine the cell populations responsible for these inflammatory signals.

 

Reviewer comment:

The reduction in inflammatory infiltrate is one of the central findings of the manuscript. However, the histological quantification method needs further clarification. The authors should specify the number of sections and fields analyzed per animal, whether field selection and image analysis were blinded, how inflammatory areas were defined, which software or thresholding method was used, and whether quantification was normalized to total tissue area. Representative images should be clearly linked to the quantitative results.

Response: We thank the reviewer for this important methodological comment. We agree that, because inflammatory infiltrate is one of the central tissue-level outcomes of the study, the histological quantification procedure required greater methodological detail.

In response, we revised the Methods section to provide a more complete description of tissue processing, image acquisition, and morphometric quantification. We now clarify that distal intestinal samples were fixed, dehydrated, cleared, paraffin embedded, sectioned at 7 µm, and stained with hematoxylin and eosin. We also specify that images were acquired at 20× magnification using a Nikon Eclipse 50i light microscope coupled to an Evolution MP 5.0 color digital camera, with image acquisition performed using Image-Pro Plus software and subsequent morphometric analysis performed in ImageJ.

We further clarified the point-counting morphometric workflow. For each animal, ten non-overlapping microscopic fields were analyzed in standardized distal intestinal sections, totaling 1,000 assessment points per animal. Field selection was performed across the intestinal muscular layer while avoiding torn, folded, or technically unsuitable areas. Histological slides, digital images, field selection, and image analysis were coded so that investigators responsible for morphometric quantification remained blinded to experimental group allocation until completion of the primary analyses.

We also defined how inflammatory areas were identified and quantified. Inflammatory infiltrate was defined as assessment points falling on inflammatory cell aggregates or inflammatory cell-rich areas within the intestinal musculature. Points corresponding to non-inflammatory tissue components, empty spaces, artifacts, or technically unsuitable regions were not counted as inflammatory infiltrate. The percentage of inflammatory infiltrate was calculated as the number of points corresponding to inflammatory infiltrate divided by the total number of valid tissue assessment points and multiplied by 100, thereby normalizing the estimate to the total tissue area evaluated through the standardized point-counting grid. We also clarified that no automated color-thresholding segmentation was used; quantification was based on blinded point-counting morphometry.

Finally, we added Supplementary Figure 1 and cited it in the Methods and Results. This figure shows the macroscopic intestinal sample preparation and representative H&E-stained sections from the T. cruzi + Saline and T. cruzi + ArtinM groups. The red arrows indicate representative areas of inflammatory infiltrate, and the overlaid point grid illustrates the structured point-counting approach used for morphometric quantification. We also clarified that these images are illustrative, whereas statistical inference was based on the blinded quantitative point-counting data obtained from all animals. These revisions improve methodological transparency and directly link the representative images to the quantitative histological endpoint.

.

 

Reviewer comment:

IL-10 did not reach significance in the univariate analysis but showed significance in the MANCOVA model after adjustment for inflammatory infiltrate. Given the small sample size and exploratory nature of this analysis, IL-10 should not be presented as a primary ArtinM-driven effect. The authors should describe the IL-10 result as exploratory or hypothesis-generating and discuss it as a possible compensatory regulatory response associated with tissue inflammation.

Response: We thank the reviewer for this important and appropriate comment. We agree that the IL-10 finding should be interpreted cautiously because IL-10 did not reach statistical significance in the primary univariate analysis and became significant only in the complementary MANCOVA model after adjustment for inflammatory infiltrate.

In response, we revised the manuscript to avoid presenting IL-10 as a primary ArtinM-driven effect. In the Results, we now clarify that IL-10 did not show significant between-group separation in the univariate analysis and should not be interpreted as a primary treatment-associated outcome. In the Discussion, we further revised the interpretation to describe the adjusted IL-10 result as secondary, exploratory, and hypothesis-generating. We also discuss IL-10 as a possible compensatory regulatory signal associated with the local inflammatory context, rather than as definitive evidence of IL-10-mediated anti-inflammatory regulation.

We additionally reinforced this caution in the limitations and conclusions, noting that the limited sample size and complementary nature of the adjusted model restrict the strength of inference regarding IL-10. These revisions ensure that the main interpretation remains focused on the primary and more robust findings: attenuation of TNF-α, IFN-γ, IL-12p40, and inflammatory infiltrate after ArtinM treatment.

 

Minor comments

Reviewer comment:

The abstract should clearly state that the scRNA-seq dataset is external and derived from chronic infection, whereas the in vivo experiment uses an acute infection model.

Response: We thank the reviewer for this important suggestion. We revised the Abstract to make this distinction explicit. The revised Abstract now states that the in vivo component was based on an acute experimental T. cruzi infection model, whereas the scRNA-seq analysis used a public murine intestinal dataset derived from a chronic infection setting.

We also clarified in the Abstract that the transcriptomic analysis was exploratory and was used only to provide pathway-level context for inflammatory mediators assessed in vivo. To avoid overinterpretation, we further stated that the public dataset was not temporally matched to the acute model and was not interpreted as a phase-matched comparator, mechanistic validation, or temporal extension of the experimental findings. This revision clearly separates the direct acute in vivo findings from the external chronic transcriptomic contextualization.

 

Reviewer comment:

The terminology should be standardized. Terms such as “validation,” “integration,” and “confirmation” should be avoided unless the authors can demonstrate direct experimental correspondence.

Response: We thank the reviewer for this important editorial and conceptual recommendation. We agree that terminology should be carefully standardized to avoid implying a level of experimental correspondence that was not directly tested.

In response, we revised the manuscript to avoid using terms such as “validation,” “confirmation,” and “integration” when referring to the relationship between the acute in vivo experiment and the external chronic scRNA-seq dataset. Throughout the Abstract, Methods, Results, Discussion, and Conclusions, we replaced stronger wording with more precise and cautious terminology, such as “exploratory pathway-level context,” “descriptive gene-module-level contextualization,” and “public scRNA-seq analysis.” We also clarified that the external transcriptomic dataset was not used as a phase-matched comparator, mechanistic validation, temporal extension, or direct confirmation of the acute ArtinM-treated model.

Where terms such as “validation” or “confirmation” remain, they are used only in a negative or limiting sense, for example to state that the scRNA-seq analysis should not be interpreted as temporal or mechanistic validation. These revisions standardize the terminology and ensure that the manuscript clearly distinguishes direct experimental findings from exploratory transcriptomic contextualization.

 

Reviewer comment:

Figure legends should provide more information on sample size, statistical tests, p-value annotations, and the composition of the inflammatory gene module.

Response: We thank the reviewer for this helpful suggestion. We revised the figure legends to improve methodological transparency and interpretability.

For the scRNA-seq figures, we added or clarified the number of cells analyzed in each public object and expanded the description of the predefined inflammatory gene module. Specifically, the Figure 2 legend now lists the complete 20-gene module used in the analysis: Tnf, Ifng, Il12a, Il12b, Il10, Tlr2, Cd14, Myd88, Nfkb1, Rela, Stat1, Ccl2, Ccl3, Ccl4, Ccl5, Cxcl9, Cxcl10, Nos2, Icam1, and Vcam1. We also clarified that the scRNA-seq visualizations were descriptive and exploratory, and that no inferential statistical comparisons were performed between the public datasets.

For the histological material, we added Supplementary Figure 1 and expanded its legend to include the number of animals per group, the number of microscopic fields and assessment points used for morphometric quantification, the statistical test applied to inflammatory infiltrate, and the relevant p-value annotations. The legend now states that inflammatory infiltrate was analyzed using the Kruskal–Wallis test followed by Dwass–Steel–Critchlow–Fligner pairwise comparisons and reports the global test result and the pairwise comparison between the T. cruzi + Saline and T. cruzi + ArtinM groups. These revisions provide clearer links between the representative images, the quantitative morphometric analysis, and the statistical results.

 

Reviewer comment:

The statistical methods should more clearly describe post hoc tests, multiple-comparison correction, MANCOVA assumptions, and covariate selection.

Response: We thank the reviewer for this important methodological suggestion. We revised the Statistical analysis section to provide a clearer description of the post hoc procedures, multiple-comparison approach, MANCOVA assumptions, and covariate selection.

Specifically, we clarified that one-way ANOVA was followed by Tukey’s post hoc test for parametric pairwise comparisons and that Tukey’s test was used as the multiple-comparison procedure after significant omnibus ANOVA results. For inflammatory infiltrate, which violated normality and homogeneity assumptions, we clarified that the Kruskal–Wallis test was followed by Dwass–Steel–Critchlow–Fligner pairwise comparisons as the nonparametric multiple-comparison procedure.

We also expanded the description of the MANCOVA assumptions. The revised text now states that homogeneity of covariance matrices was assessed using Box’s M test and that multivariate normality was evaluated using the multivariate Shapiro–Wilk test. In addition, we clarified that the multivariate effect was assessed using Pillai’s trace, Wilks’ lambda, Hotelling’s trace, and Roy’s largest root.

Finally, we clarified the rationale for covariate selection. Inflammatory infiltrate was selected a priori as the covariate because it was the primary histological endpoint and represents local tissue inflammatory burden, which could influence tissue cytokine concentrations. We also specified that the adjusted univariate follow-up tests from the MANCOVA were interpreted as complementary to the primary univariate analyses and not as the sole basis for mechanistic inference. These revisions improve the transparency and reproducibility of the statistical workflow.

 

Reviewer comment:

The animal methods would benefit from additional details on randomization, blinding, humane endpoints, body weight monitoring, mortality, and clinical signs.

Response: We thank the reviewer for this important methodological recommendation. We agree that additional details regarding animal allocation, monitoring, welfare procedures, and blinding improve transparency and reproducibility.

In response, we expanded the animal methods section. We now clarify that group allocation was performed before treatment initiation using a computer-generated random sequence. We also added that cage positions were balanced to reduce rack-location effects, husbandry was standardized across groups, and handling, dosing, and tissue collection order were alternated within a consistent daily time window to reduce circadian and batch-related variation.

We also expanded the description of animal monitoring and humane endpoints. The revised manuscript now states that animals were monitored daily for posture, grooming, mobility, feeding behavior, and body weight variation. Humane endpoints were predefined and included persistent anorexia, marked lethargy, severe weight loss, or inability to access food or water. We further clarified that no unexpected adverse events, premature euthanasia, or data exclusions occurred during the experiment.

Finally, we clarified the blinding procedures. Tissue samples, histological slides, ELISA plates, and image files were labeled with anonymized codes, and investigators responsible for morphometric quantification, ELISA readout, and statistical analysis remained blinded to group allocation until completion of the primary analyses. These revisions provide a more complete description of randomization, blinding, monitoring, clinical assessment, mortality-related endpoints, and animal welfare procedures.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the correction and attention to the comments. Good luck in your further research.

Reviewer 4 Report

Comments and Suggestions for Authors

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Reviewer 5 Report

Comments and Suggestions for Authors

Authors have replied all comments accordingly.