Strain-Specific microRNA Reprogramming of Human Dendritic Cells by Probiotic and Commensal Escherichia coli Outer Membrane Vesicles

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

This revised manuscript investigates how outer membrane vesicles (OMVs) from a probiotic E. coli strain (EcN) and commensal strains (ECOR12/ECOR63) shape human monocyte-derived dendritic cell (Mo-DC) maturation, cytokine release, and a targeted miRNA panel, using a paired donor design and multivariate analyses to define strain-specific immune “fingerprints”. Overall, the revision is improved, clearer in framing the core integrated arm vs. the extended phenotyping arm, and the conclusions are better aligned with the available dataset.

Abstract

1. Clarify the key take-home in one sentence and reduce density. The abstract is informative but long and packed with technical detail; consider shortening and making the primary conclusion more direct (what is the most important biological message?). (Abstract, p.1, lines 12–25)
2. Good clarification of ECOR63 scope. The statement that ECOR63 was excluded from miRNA/integrated PCA due to a missing matched miRNA is appropriate and should remain explicit. (Abstract, p.1, lines 23–25)

Introduction

1. Strong background but slightly repetitive. The first paragraphs provide solid context, but some concepts are restated (microbiota/immune balance and “tractable inputs”). Consider tightening for readability. (Introduction, p.1, lines 31–40; p.2, lines 41–58)
2. Figures 1–2: ensure permissions/originality are compliant and readable. Please confirm the figures are author-generated or appropriately licensed, and ensure all labels are legible at the journal layout size. (Fig. 1 caption, p.2, lines 59–64; Fig. 2 caption, p.3, lines 90–94)

Materials and Methods

1. Study design is now clearly described, good. The separation between the core integrated arm and extended phenotyping arm is clearly explained and well summarized in Table 1. (2.1 Experimental design, p.7, lines 233–241; Table 1, p.7, lines 242–249)
2. OMV isolation: report centrifugation in ×g (not only rpm). Ultracentrifugation is described with rpm; for reproducibility, please provide RCF (×g) and rotor type, because rpm is rotor-dependent. (2.4 OMV production/isolation, p.9, lines 299–314)
3. OMV input normalization: protein-only normalization is acceptable, but add/clarify QC. Since dose is standardized by total protein, please add (even briefly) whether you assessed particle concentration/size distribution (e.g., NTA/DLS) or endotoxin/LPS variability, or clearly state this as not performed. (You discuss this limitation later, but adding a short note in Methods improves transparency.) (Table 2 OMV input control, p.8, lines 262–264; OMV isolation/quantification, p.9, lines 304–306)
4. Flow cytometry: add a short gating/QC description. Please specify the basic gating strategy (singlets/live cells/monocytes/DC gate), compensation controls, and whether fluorescence is reported as % positive and/or MFI (and where). (2.7 Flow cytometry, p.10, lines 328–335; Table 3, p.9, lines 324–325)
5. RT-qPCR: clarify reference/normalizer selection. You mention UNISP and housekeeping/reference targets; please specify which reference(s) were ultimately used for normalization across donors/conditions (or add this in a supplement). (2.9 miRNA extraction/RT-qPCR, p.10, lines 348–352)
6. Statistics: overall appropriate; consider adding exact software/version and handling of missingness/outliers. The workflow is clear; adding software versions and stating how outliers/missing values were handled would strengthen reproducibility. (2.10–2.11 Data analysis/hypotheses, p.10, lines 358–365; p.11, lines 366–392; Table 4, p.11, lines 377–379)

Results

1. Table 5 is not very informative in its current form. A table showing volumes to reach a 20 µL final mixture adds limited scientific value; consider replacing it with more informative yield metrics (e.g., protein yield per culture volume, particles/mL, or a summary of replicate preparations), or move it to Supplementary. (3.1, p.12, lines 405–409; Table 5, p.12, lines 407–409)
2. Cryo-TEM figure: add minimal quantification/replication statement. The images are helpful; please state how many preparations/fields were examined and whether the size distribution was consistent across replicates (even qualitative). (Fig. 5 and text, p.12, lines 410–418)
3. SDS-PAGE figure quality needs improvement. The gel image appears low-resolution, with a heavy background and overlaid arrows/text; please replace with a higher-quality original scan, use consistent English labeling (“Marker” instead of “Marcador”), and ensure that lane/band visibility and contrast are adequate. (3.1.1 + Fig. 6, p.13, lines 419–428)

Discussion / Limitations / Conclusions

1. Good and appropriately cautious discussion of protein normalization limitations. This section is well written and balanced. (Discussion, p.20, lines 592–599)
2. Limitations/Future Directions are strong—keep them. Clear, relevant, and aligned with what the study can and cannot claim. (Section 5, p.25, lines 825–845)
3. Conclusions are much better framed, but keep wording conservative. The separation of “core integrated dataset” conclusions from the ECOR63 phenotyping context is appropriate; continue to avoid causal/mechanistic overstatement beyond the data shown. (Section 6, p.25, lines 846–865)

English editing comments

Overall, the English is readable, but several places would benefit from minor editing for clarity, grammar, and style consistency. Below are specific, line-located examples with suggested fixes.

Abstract

• p.1, lines 12–15: First sentence is long and slightly dense. Consider splitting for clarity.

Example fix: “Outer membrane vesicles (OMVs) mediate microbe–host communication. However, whether probiotic and commensal E. coli OMVs program Mo-DCs through distinct cytokine–miRNA circuits remains unclear.”

• p.1, lines 22–25: The parenthetical clause is heavy. Consider rephrasing more directly.

Example fix: “…ECOR63 OMVs were assessed by ELISA and flow cytometry; however, miRNA profiling and integrated PCA were not performed because matched miRNA data were unavailable.”

Introduction

• p.2, lines 41–44: Missing punctuation/structure makes the sentence awkward: “defined, isolatable microbial output outer membrane vesicles…”

Fix: “…by focusing on a defined, isolatable microbial output: outer membrane vesicles (OMVs)…”

• p.2, lines 53–54: If written as “lumenal”, use the more standard “luminal” (common in biomedical writing).
• p.2, lines 56–58: Phrase “experimentally tractable microbial inputs capable of modulating…” is correct but wordy.

Tighten: “We use this framework to motivate OMVs as tractable microbial inputs that modulate the epithelial–immune axis.”

Objectives / Transition to Methods

• p.7, lines 222–229: The objectives sentence is very long (multiple lists inside lists).

Fix: Split into 2–3 sentences and keep parallel verbs (e.g., “characterize…”, “contextualize…”, “apply…”).

Methods

• p.9, lines 297–306: Several steps are packed into one long paragraph; readability improves if broken into shorter sentences (production → clarification/filtration → concentration → ultracentrifugation → resuspension/storage).

Also consider simplifying phrases like “under cold chain conditions” to “kept at 4 °C/on ice” for clarity.

Results

• p.12, lines 395–399: Tense consistency: “hypotheses and inferential statistics… apply” → better as “were applied” (since this is describing what you did).

Fix: “All a priori hypotheses and inferential statistics… were applied to the core integrated dataset…”

• p.12, lines 415–418: Hyphenation/style: use “double-membrane” (not “double membrane”) and consider “typically ranged” instead of “typically range” for tense consistency with the paragraph.
• p.13, Figure 6 label / p.13 lines 425–428: The gel lane label “Marcador” should be in English (“Marker”). Also, ensure consistent terminology: “band masses” → better “molecular weights”.
• p.13, lines 431–432: Hyphenation: “strain dependent” → “strain-dependent”.

Discussion

• p.20, lines 579–581: Grammar/punctuation issue:

“supported the expected morphology of OMVs, rounded vesicles…”

Fix: “supported the expected morphology of OMVs: rounded vesicles with…”

General consistency (throughout)

• Use consistent hyphenation for compound modifiers: “microbiota-derived,” “strain-specific,” “donor-blocked,” “protein-based,” “paired within-donor”.
• Watch for very long sentences with multiple parentheses; splitting often improves clarity without changing meaning.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Given that this is a resubmitted manuscript and the authors have made all the recommended corrections, I can only repeat the general conclusion of the peer-reviewed article and recommend it for publication.

The manuscript under review examines the properties of OMV from probiotic and commensal Escherichia coli strains. The authors provide a comprehensive rationale for the study’s relevance by systematizing the data in figures and tables. The “Materials and Methods” section provides sufficient detail, specifying all the methods, reagents, and instruments used. Notably, the authors separately systematized all statistical methods used in a table. The results are presented in detail, and their reliability is confirmed by figures and tables. The authors first substantiated the qualitative and quantitative preparation of OMV preparations and then demonstrated the specific results obtained using DC stimulation. Furthermore, given that OMV stimulation triggers reaction chains, multivariate models were used to visualize the obtained data. The Discussion section also fully explores the main and conceptual questions the authors posed for the purposes of their study. Finally, the authors highlight the key limitations and prospects of their study and summarize the main directions in their conclusion.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Thank you for the careful revision. The updated manuscript addresses the key issues raised in the previous review by clarifying the study structure into a core integrated arm and an extended phenotyping arm, resolving the earlier inconsistency in strain inclusion (particularly the handling of ECOR63) and documenting it clearly (e.g., assay coverage by strain). You also strengthened transparency and reproducibility by adding complete human ethics/consent information and donor details, and by explicitly defining biological replication (donors) versus technical replicates to avoid pseudo-replication, including a clear explanation of how “n” was derived across analyses. In addition, the flow-cytometry antibody/fluorochrome labeling is now presented consistently between Methods and Results. Overall, these revisions substantially improve methodological clarity, compliance, and interpretability of the findings.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript under review examines the properties of OMV from probiotic and commensal Escherichia coli strains. The authors provide a comprehensive rationale for the study’s relevance by systematizing the data in figures and tables. The “Materials and Methods” section provides sufficient detail, specifying all the methods, reagents, and instruments used. Notably, the authors separately systematized all statistical methods used in a table. The results are presented in detail, and their reliability is confirmed by figures and tables. The authors first substantiated the qualitative and quantitative preparation of OMV preparations and then demonstrated the specific results obtained using DC stimulation. Furthermore, given that OMV stimulation triggers reaction chains, multivariate models were used to visualize the obtained data. The Discussion section also fully explores the main and conceptual questions the authors posed for the purposes of their study. Finally, the authors highlight the key limitations and prospects of their study and summarize the main directions in their conclusion. I would like to make the following minor comments:
1. L. 371-373: The a, b, and c designations should be added to the figure itself. They are in the caption, but not in the figure.
2. L. 404-406: Add the * and # designations to the figure caption.
3. Section 3.2.2. DC maturation by flow cytometry: Agree: the text in Section 3.2.2 (L. 414-415), the information in Figure 8 (L. 418), and the caption to Figure 8 (L. 419-421). Figure 8 shows the flow cytometry data for DCs treated with OMV ECOR63. Add this information to the text of the results and the figure caption or remove the data from the figure.

Author Response

For research article

 

Response to Reviewer 1 Comments
1. Summary
Thank you very much for taking the time to review our manuscript. Your comments were extremely helpful and have substantially improved the clarity, methodological transparency, and overall rigor of the study. We have carefully addressed each point raised and revised the manuscript accordingly. All changes have been incorporated in the revised version and are highlighted in the resubmitted files, and detailed point-by-point responses are provided below.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes	The Introduction has been refined to strengthen the mechanistic framing of OMV–host communication and dendritic-cell programming, and to clearly motivate the selected miRNA panel (miR-155, let-7i, miR-146b, miR-29a) through explicit linkage to PRR/TLR signaling and downstream NF-κB/AP-1 regulatory circuits. Background text was tightened to maintain focus on the study’s central hypothesis and endpoints.
Are all the cited references relevant to the research?	Yes	All cited references are directly relevant to the research question.
Is the research design appropriate?	Yes	The experimental design was unified and clarified to eliminate strain-related ambiguity by explicitly defining a two-tier structure: a core integrated arm (iDC control, EcN OMVs, ECOR12 OMVs) used for cytokines, flow cytometry, miRNA RT-qPCR, and integrated multivariate analyses (N = 20 donors; n = 60 donor–condition observations), and an extended phenotyping arm that additionally includes ECOR63 for ELISA and flow cytometry (and OMV profiling) (N = 20 donors; n = 80). The manuscript now states that ECOR63 is not included in miRNA profiling or integrated PCA/MANOVA due to unavailable matched miRNA measurements, ensuring full transparency and reproducibility across sections.
Are the methods adequately described?	Yes	The Methods section was expanded and reorganized to improve reproducibility, including: (i) explicit ethics/IRB approval, informed consent, and donor inclusion/exclusion criteria; (ii) a clear definition of biological replication (N donors) vs technical triplicates and how triplicates were aggregated; (iii) standardized and internally consistent flow-cytometry antibody/fluorochrome assignments; and (iv) a detailed statistical framework that treats donors as the repeated-measures subject/block (paired design), preventing pseudo-replication and clarifying the unit of inference.
Are the results clearly presented?	Yes	The Results were revised to align strictly with the declared study arms and assay coverage. ECOR63-related findings are presented only within the extended phenotyping outputs (ELISA/flow/OMV profiling), while miRNA results and integrated multivariate analyses are restricted to the core dataset (Control/EcN/ECOR12). Figure legends and text now explicitly report sample sizes, define what constitutes an observation, and describe the statistical approach used for within-donor comparisons, improving clarity and interpretability.
Are the conclusions supported by the results?	Yes	The Conclusions were tightened to match the evidence hierarchy: strain-specific cytokine–miRNA “fingerprints” are claimed only for the core integrated dataset (Control/EcN/ECOR12), while ECOR63 is discussed as contextual phenotyping support (ELISA/flow/OMV profiling) rather than as part of the integrated miRNA-based fingerprint. Limitations and future directions were strengthened to emphasize that definitive DC polarization/functional outcomes require expanded marker panels and functional assays (e.g., costimulatory markers, CCR7, T-cell priming co-cultures), ensuring conclusions remain fully supported by the presented data.
3. Point-by-point response to Comments and Suggestions for Authors
Comment 1: L. 371-373: The a, b, and c designations should be added to the figure itself. They are in the caption, but not in the figure.
Response 1: Thank you for this observation. We agree that panel labels must be consistent between the figure and its caption to avoid confusion. After careful revision, we determined that the a/b/c designations were unnecessary because the figure is presented as a single composite image rather than as distinct subpanels. Therefore, we removed the a/b/c labels from the caption and revised the caption text to describe the figure content clearly without panel references. This change can be found on page 10, lines 445–447 (Figure 5 caption).
Comment 2: L. 404-406: Add the * and # designations to the figure caption. Response 2: Thank you. We agree that all statistical symbols used in the figure should be explicitly defined in the caption. We have updated the figure caption to describe the meaning of the * and # symbols, including the reference groups, statistical test, repeated-measures design (donor as subject), multiple-comparison adjustment, and significance threshold. This clarification improves interpretability and transparency. The revised caption can be found on page 11, lines 483–490 (Figure 7 caption).
Comment 3: Section 3.2.2. DC maturation by flow cytometry: Agree: the text in Section 3.2.2 (L. 414-415), the information in Figure 8 (L. 418), and the caption to Figure 8 (L. 419-421). Figure 8 shows the flow cytometry data for DCs treated with OMV ECOR63. Add this information to the text of the results and the figure caption or remove the data from the figure.
Response 3: Thank you for highlighting this inconsistency. We agree that the Results text, figure content, and caption must be fully aligned. We have revised Section 3.2.2 to explicitly state that Mo-DCs were stimulated with OMVs from EcN, ECOR12, and ECOR63, in addition to iDC controls, and updated the Figure 8 caption accordingly. This ensures that ECOR63 is consistently described across the text and figure, improving clarity and reproducibility. These changes can be found on page 12, lines 497–501 (Section 3.2.2) and page 12, lines 503–506 (Figure 8 caption).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript examines whether outer membrane vesicles (OMVs) derived from probiotic E. coli Nissle 1917 and commensal E. coli strains differentially program human monocyte-derived dendritic cells, integrating DC maturation markers and cytokine secretion with miRNA signatures and multivariate analyses to define strain-specific immune “fingerprints.” 

1. A) Abstract

1. Strain inconsistency already visible in the main paper structure: later, the experimental design and Table 1 list only EcN and ECOR12 as exposure groups, but the Results/Discussion repeatedly interpret ECOR63 as well (see details below). This creates a major clarity and reproducibility issue that should be explicitly resolved in the Abstract (either remove ECOR63 from the story or add ECOR63 consistently across Methods/Results/analyses).
• Example of mismatch in Results section: cytokines are explicitly compared across EcN, ECOR12, and ECOR63 (Lines 401–403)

• But flow cytometry and miRNA later use only EcN and ECOR12 (Lines 413–416, 425–428). Abstract must reflect what was actually done consistently.

1. Abstract should report experimental replication details (e.g., number of donors/biological replicates and how “n” is defined), because later multivariate analysis claims n = 60 (Figure 11 legend, Lines 456–457) without clarifying what constitutes an independent sample.

B) Introduction

1. Good broad background, but the manuscript should sharpen the specific mechanistic hypothesis about why these particular miRNAs (miR-155, let-7i, miR-146b, miR-29a) are expected to respond differently by strain, beyond citing “key regulatory layers.” The rationale is mentioned, but the expected directionality and link to PRR/TLR signaling could be stated more explicitly before Methods.
2. The rationale for strain selection needs clarity: the objectives refer broadly to “commensal strains,” but the Methods/Design later specify only ECOR12 as the commensal comparator. This should be reconciled in the Introduction framing so readers understand whether ECOR63 is part of the main experimental design or only a partial add-on.

C) Materials and Methods

C1) Experimental design/study groups

1. Major design inconsistency: the design statement says the comparative design tests OMVs from EcN vs ECOR12 (Lines 244–247)—no ECOR63 is included here.
2. Table 1 exposure groups include only EcN and ECOR12 (Lines 254–256):
• “Exposure groups: (i) Unstimulated iDC control; (ii) iDC + EcN OMVs; (iii) iDC + ECOR12 OMVs. Yet the Results include ECOR63 in cytokines and OMV protein profiling (see Results). Either (a) add ECOR63 into experimental groups and all downstream assays, OR (b) remove ECOR63 from Results/Discussion claims, OR (c) clearly label ECOR63 work as a separate exploratory arm and adjust statistics accordingly.

C2) Human samples/ethics reporting gap (major)

1. The Methods state that peripheral blood from healthy donors was used (Lines 270–272), but no ethics approval / informed consent / IRB statement is visible in this section. Add explicit ethics committee approval ID, consent procedure, and compliance statement (Declaration of Helsinki), and include donor inclusion/exclusion and number of donors.

C3) Flow cytometry methods mismatch vs Results (major)

1. Antibody fluorochrome assignment mismatch:
• Methods equipment table lists: anti-CD14-FITC, anti-CD83-PE, anti-CD209-APC (Lines 6–7)
• Results flow staining describes anti-CD14-FITC, anti-CD209-PE, and anti-CD83-APC (Lines 415–416). Correct the inconsistency; this directly affects reproducibility and figure interpretation.

C4) Replication/statistics transparency

1. Table 1 says “independent experiments with technical processing in triplicate” (Line 254) but doesn’t define how many donor-derived Mo-DC preparations or biological replicates were used. Provide N donors, N independent DC differentiations, and whether stats treat donors as random effects.

D) Results (major issues by subsection)

D1) OMV characterization

1. Protein profiling explicitly compares EcN, ECOR12, and ECOR63 (Lines 388–391). ECOR63 appears here but is not included in the stated Methods design/groups (see Table 1 and design lines 244–247). This suggests either incomplete coverage of Methods or an inconsistent experimental design.

D2) Cytokine analysis (ELISA)

1. Cytokines are compared across EcN, ECOR12, ECOR63 (Lines 401–403)
2. Key finding claims are clear, but interpretation depends on consistent strain inclusion:
• ECOR12 higher IL-10 vs EcN and ECOR63 (Lines 407–409)
• ECOR63 has the highest IL-6 and TNF-α (Lines 409–411). If ECOR63 is truly included, then ECOR63 must also appear in Methods and statistical hypotheses and ideally flow/miRNA assays.

D3) Flow cytometry maturation

1. Flow cytometry is performed only with EcN and ECOR12 (Lines 413–416). This prevents linking the ECOR63 cytokine phenotype to the maturation marker phenotype, thereby weakening strain-comparative conclusions.

D4) miRNA results

1. miRNA assays are performed only with EcN and ECOR12 (Lines 425–428). Again, ECOR63 is absent—yet later the Discussion interprets ECOR63 as a distinct immunological “tone.” This should be either tested or removed/softened.

D5) PCA / multivariate integration

1. PCA integrates cytokines and miRNA and reports n = 60, but the shape legend includes only Control, EcN, and ECOR12 (Lines 456–457). PCA omits ECOR63 even though the cytokine ELISA includes it. This makes the multivariate “strain separation” claim incomplete; either include ECOR63 across all modalities, or explicitly state that PCA is only ECN vs ECOR12 vs control.

E) Discussion

1. The Discussion interprets strain-specific “tone” and explicitly contrasts ECOR12 vs ECOR63 (Lines 589–590). This is not fully supported because ECOR63 is not included in flow cytometry/miRNA experiments (Results lines 413–428). Either add ECOR63 to those assays or limit ECOR63 claims to cytokines/protein profiling only.
2. The manuscript appropriately notes that DC polarization cannot be inferred from a single marker (Lines 602–605). Still, this argument should also motivate a more complete phenotype panel or functional readouts if claiming differential polarization.

F) Conclusions

1. The Conclusions state broad strain-comparative claims; however, given that ECOR63 is not consistently included across assays and statistical frameworks, the conclusions should be tightened to match the actual datasets (cytokines vs flow/miRNA vs multivariate).
• The manuscript itself summarizes that probiotic and commensal strains yield distinct fingerprints (Conclusions area, Lines 776–781). Rephrase conclusions to reflect what is supported across all platforms, and clearly separate “supported by full multi-omic panel” vs “supported only by ELISA/protein profiling.”ِ

Comments on the Quality of English Language

Overall, the manuscript is readable, and the scientific meaning is generally clear; however, the English should be improved to enhance clarity and professionalism. The main issues are (i) frequent mid-word hyphenation/word breaks that disrupt readability, (ii) very long sentences (especially in the Abstract) that would benefit from splitting, and (iii) consistency issues in terminology and spelling (e.g., signaling vs signalling; microRNA vs miRNA).

1) Frequent mid-word hyphenation / broken words (major readability problem)

These appear throughout and should be removed in the clean manuscript (often caused by manual hyphenation or formatting export):

• Abstract: “candi-dates”, “den-dritic”, “monocyte-de-rived”, “matu-ration”, “strain-en-coded”, “mi-croRNAs” (Lines 12–37). 
• Methods: “fil-trate”, “swing-ing-bucket”, “ultracentri-fu-gation”, “cryogen-ic”, “phys-iologically” (Lines 283–287). 
• Results: “matu-ration”, “ex-posed”, “re-lease”, “evalu-ate” (Lines 395–401, 407–410, 426–428). 
• Discussion/late section: “inter-pretable”, “clar-ify”, “pat-tern”, “signifi-cance”, “ade-quacy”, “strat-egy”, “engi-neered” (Lines 721–752).   

Suggestion: Ensure the editable source file (Word/LaTeX) has no manual hyphen characters inserted mid-word; re-export to PDF cleanly.

2) Overlong, multi-clause sentences (reduce clarity; split into shorter sentences)

• Abstract contains several very long sentences with many clauses and statistics in the same line block (Lines 14–36).  Split into 4–5 shorter sentences: (background) → (what you did) → (key cytokine result) → (key miRNA result) → (statistics + conclusion).

3) Consistency in spelling (choose one style and apply everywhere)

• The Abstract uses “TLR signalling” (British spelling) (Line 21) while elsewhere the manuscript uses “signaling” in the Methods section (Lines 334–336). Decide on American English (signaling) or British English (signalling) per journal preference and apply consistently.

4) Terminology consistency (avoid switching forms within the same passage)

• In the Abstract, both “microRNAs (miRNAs)” and later “miRNAs” are used interchangeably; also, the keywords list shows “mi-croRNAs” due to formatting (Lines 14–30).  Define once (“microRNAs, miRNAs”), then use miRNA/miRNAs consistently.

 

5) Minor style/clarity polishing (tighten phrasing)

Examples where minor edits would improve clarity and flow:

• “DC maturation was evaluated using two complementary approaches…” is clear, but later wording becomes repetitive (“quantifications are shown…”, “results are shown…”) (Lines 394–417). 

Suggestion: Vary structure and reduce “shown” repetition (e.g., “Figure X summarizes…”).

Author Response

For research article

 

Response to Reviewer 2 Comments
1. Summary
Thank you very much for taking the time to review our manuscript. Your comments were extremely helpful and have substantially improved the clarity, methodological transparency, and overall rigor of the study. We have carefully addressed each point raised and revised the manuscript accordingly. All changes have been incorporated in the revised version and are highlighted in the resubmitted files, and detailed point-by-point responses are provided below.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Can be improved	The Introduction has been refined to strengthen the mechanistic framing of OMV–host communication and dendritic-cell programming, and to clearly motivate the selected miRNA panel (miR-155, let-7i, miR-146b, miR-29a) through explicit linkage to PRR/TLR signaling and downstream NF-κB/AP-1 regulatory circuits. Background text was tightened to maintain focus on the study’s central hypothesis and endpoints.
Are all the cited references relevant to the research?	Must be improved	All cited references are directly relevant to the research question.
Is the research design appropriate?	Must be improved	The experimental design was unified and clarified to eliminate strain-related ambiguity by explicitly defining a two-tier structure: a core integrated arm (iDC control, EcN OMVs, ECOR12 OMVs) used for cytokines, flow cytometry, miRNA RT-qPCR, and integrated multivariate analyses (N = 20 donors; n = 60 donor–condition observations), and an extended phenotyping arm that additionally includes ECOR63 for ELISA and flow cytometry (and OMV profiling) (N = 20 donors; n = 80). The manuscript now states that ECOR63 is not included in miRNA profiling or integrated PCA/MANOVA due to unavailable matched miRNA measurements, ensuring full transparency and reproducibility across sections.
Are the methods adequately described?	Can be improved	The Methods section was expanded and reorganized to improve reproducibility, including: (i) explicit ethics/IRB approval, informed consent, and donor inclusion/exclusion criteria; (ii) a clear definition of biological replication (N donors) vs technical triplicates and how triplicates were aggregated; (iii) standardized and internally consistent flow-cytometry antibody/fluorochrome assignments; and (iv) a detailed statistical framework that treats donors as the repeated-measures subject/block (paired design), preventing pseudo-replication and clarifying the unit of inference.
Are the results clearly presented?	Can be improved	The Results were revised to align strictly with the declared study arms and assay coverage. ECOR63-related findings are presented only within the extended phenotyping outputs (ELISA/flow/OMV profiling), while miRNA results and integrated multivariate analyses are restricted to the core dataset (Control/EcN/ECOR12). Figure legends and text now explicitly report sample sizes, define what constitutes an observation, and describe the statistical approach used for within-donor comparisons, improving clarity and interpretability.
Are the conclusions supported by the results?	Can be improved	The Conclusions were tightened to match the evidence hierarchy: strain-specific cytokine–miRNA “fingerprints” are claimed only for the core integrated dataset (Control/EcN/ECOR12), while ECOR63 is discussed as contextual phenotyping support (ELISA/flow/OMV profiling) rather than as part of the integrated miRNA-based fingerprint. Limitations and future directions were strengthened to emphasize that definitive DC polarization/functional outcomes require expanded marker panels and functional assays (e.g., costimulatory markers, CCR7, T-cell priming co-cultures), ensuring conclusions remain fully supported by the presented data.
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Strain inconsistency already visible in the main paper structure: later, the experimental design and Table 1 list only EcN and ECOR12 as exposure groups, but the Results/Discussion repeatedly interpret ECOR63 as well (see details below). This creates a major clarity and reproducibility issue that should be explicitly resolved in the Abstract (either remove ECOR63 from the story or add ECOR63 consistently across Methods/Results/analyses).
Response 1: Thank you for pointing this out. We agree this inconsistency affected clarity and reproducibility. Therefore, we revised the Abstract to explicitly distinguish the core integrated arm (iDC control, EcN OMVs, ECOR12 OMVs) from the extended phenotyping arm (adding ECOR63 OMVs for ELISA/flow cytometry only), and to state that ECOR63 was not included in miRNA profiling and integrated multivariate analysis due to the lack of matched miRNA measurements. This change can be found – page 1, lines 11–26 (Abstract, highlighted).
Comments 2: Abstract should report experimental replication details (e.g., number of donors/biological replicates and how “n” is defined), because later multivariate analysis claims n = 60 (Figure 11 legend, Lines 456–457) without clarifying what constitutes an independent sample. Response 2: Thank you. We agree replication and “n” definition must be transparent. We added explicit replication language in the Abstract stating N = 20 independent healthy donors, with a paired within-donor design, and clarified how donor-condition observations relate to n in integrated analyses. This change can be found – page 1, lines 13–19 (Abstract). In addition, we clarified the unit of inference and donor-blocked framework in the Statistics section – page 11, lines 392–410 (Statistical analysis; donor as subject/block; paired contrasts; avoidance of pseudo-replication).
Comments 3: Good broad background, but the manuscript should sharpen the specific mechanistic hypothesis about why these particular miRNAs (miR-155, let-7i, miR-146b, miR-29a) are expected to respond differently by strain, beyond citing “key regulatory layers.” The rationale is mentioned, but the expected directionality and link to PRR/TLR signaling could be stated more explicitly before Methods.
Response 3: Thank you. We agree that the mechanistic hypothesis needed clearer directionality and explicit linkage to PRR/TLR signaling. We revised the Introduction to state expected strain-dependent PRR/TLR-driven programs and the corresponding anticipated miRNA directionality (e.g., pro-inflammatory vs regulatory programs). This change can be found – page 4, lines ~136–144 (highlighted mechanistic hypothesis paragraph referencing TLR4 and miRNA regulatory circuits).
Comments 4: The rationale for strain selection needs clarity: the objectives refer broadly to “commensal strains,” but the Methods/Design later specify only ECOR12 as the commensal comparator. This should be reconciled in the Introduction framing so readers understand whether ECOR63 is part of the main experimental design or only a partial add-on.
Response 4: Thank you. We agree and revised the strain-selection framing to define ECOR12 as the pre-specified commensal comparator in the core integrated arm, and ECOR63 as an additional commensal reference included only in an extended phenotyping arm (ELISA/flow cytometry/OMV profiling), clearly separated from miRNA and integrated multivariate analyses. This change can be found – page 6, lines ~217–233 (highlighted paragraph clarifying “core” vs “extended phenotyping” arms and assay coverage).
Comments 5: Major design inconsistency: the design statement says the comparative design tests OMVs from EcN vs ECOR12 (Lines 244–247)—no ECOR63 is included here.
Response 5: Thank you. We addressed this by rewriting the Experimental Design section to explicitly define: (i) a core integrated arm (control, EcN, ECOR12) used for cytokines + flow + miRNA + integrated multivariate analyses; and (ii) an extended phenotyping arm that adds ECOR63 for cytokine ELISA and flow cytometry (and OMV profiling), while keeping integrated miRNA/multivariate analyses restricted to the core arm due to unmatched miRNA availability. This change can be found – page 8, lines ~264–285 (Experimental design text) and Table 1 on page 8, lines ~275–286 (assay-by-group matrix and notes).   Comments 6: Table 1 exposure groups include only EcN and ECOR12 (Lines 254–256): • “Exposure groups: (i) Unstimulated iDC control; (ii) iDC + EcN OMVs; (iii) iDC + ECOR12 OMVs. Yet the Results include ECOR63 in cytokines and OMV protein profiling (see Results). Either (a) add ECOR63 into experimental groups and all downstream assays, OR (b) remove ECOR63 from Results/Discussion claims, OR (c) clearly label ECOR63 work as a separate exploratory arm and adjust statistics accordingly. Response 6: Thank you. We followed option (c) and made the design consistent across the manuscript by explicitly labeling ECOR63 as part of an extended phenotyping arm and aligning Methods, Results, and Discussion accordingly. Table 1 was revised to show assay coverage (core vs extended), and the Statistics section now distinguishes the inferential framework for core integrated analyses vs extended phenotyping comparisons. These changes can be found – page 8, lines ~264–285 (Design + Table 1) and page 11, lines ~392–409 (Statistical analysis framework).   Comments 7: The Methods state that peripheral blood from healthy donors was used (Lines 270–272), but no ethics approval / informed consent / IRB statement is visible in this section. Add explicit ethics committee approval ID, consent procedure, and compliance statement (Declaration of Helsinki), and include donor inclusion/exclusion and number of donors. Response 7: Thank you. We agree this was a major omission. We added a full ethics/IRB statement, Declaration of Helsinki compliance, written informed consent, the number of donors (N = 20), and explicit inclusion/exclusion criteria suitable for “healthy donor” recruitment. The ethics approval is now specified as: Bioethics 631 Committee of the University of Barcelona (Institutional Review Board: 1R800003099), 2020. This change can be found – page 9, lines ~318–329 (ethics approval, consent, inclusion/exclusion criteria, anonymization).   Comments 8: Antibody fluorochrome assignment mismatch: • Methods equipment table lists: anti-CD14-FITC, anti-CD83-PE, anti-CD209-APC (Lines 6–7) • Results flow staining describes anti-CD14-FITC, anti-CD209-PE, and anti-CD83-APC (Lines 415–416). Correct the inconsistency; this directly affects reproducibility and figure interpretation. Response 8: Thank you. We agree this required correction for reproducibility. We standardized the fluorochrome assignments and ensured the Results text matches the Methods equipment table: anti-CD14-FITC, anti-CD209-APC, and anti-CD83-PE. This change can be found – page 15-16, lines ~498–506 (Results flow cytometry staining description) and the acquisition/settings table remains consistent – page 10, lines ~358–360 (Flow cytometer/antibody listing).   Comments 9: Table 1 says “independent experiments with technical processing in triplicate” (Line 254) but doesn’t define how many donor-derived Mo-DC preparations or biological replicates were used. Provide N donors, N independent DC differentiations, and whether stats treat donors as random effects. Response 9: Thank you. We clarified that Mo-DCs were generated from N = 20 independent donors, where each donor constitutes an independent Mo-DC differentiation, and that conditions were applied in a paired within-donor design. We also specified that the donor is treated as the subject/block in repeated-measures / donor-blocked inference (and, where applicable, framed as a donor random intercept conceptually for controlling inter-donor variability). These changes can be found – page 9, lines ~299–317 (donors and paired design) and page 11, lines ~392–410 (donor-blocked MANOVA / repeated-measures ANOVA; prevention of pseudo-replication; paired post-hoc contrasts).   Comments 10: Protein profiling explicitly compares EcN, ECOR12, and ECOR63 (Lines 388–391). ECOR63 appears here but is not included in the stated Methods design/groups (see Table 1 and design lines 244–247). This suggests either incomplete coverage of Methods or an inconsistent experimental design. Response 10: Thank you. We resolved this by explicitly placing protein profiling within the extended phenotyping arm and reflecting this in Experimental Design + Table 1 (assay coverage matrix). The Results text now aligns with that structure and frames ECOR63 outputs as extended phenotyping context. This change can be found – page 8, Table 1 and notes (lines ~275–285) and the aligned Results section – page 14, lines ~464–466 (protein profile comparison including ECOR63 is explicitly contextualized).   Comments 11: Cytokines are compared across EcN, ECOR12, ECOR63 (Lines 401–403). Response 11: Thank you. This has been retained but is now explicitly labeled as the extended phenotyping arm (control, EcN, ECOR12, ECOR63), with donor replication and donor-blocked statistics clearly described. This change can be found – page 15, Figure 7 legend and associated text, lines ~477–490, where the extended phenotyping dataset and donor-condition counts are stated.   Comments 12: Key finding claims are clear, but interpretation depends on consistent strain inclusion: • ECOR12 higher IL-10 vs EcN and ECOR63 (Lines 407–409) • ECOR63 has the highest IL-6 and TNF-α (Lines 409–411). If ECOR63 is truly included, then ECOR63 must also appear in Methods and statistical hypotheses and ideally flow/miRNA assays. Response 12: Thank you. We ensured ECOR63 inclusion is consistent across the assays where it was performed: ELISA + flow cytometry + OMV protein profiling (extended phenotyping arm), and we explicitly state that ECOR63 was not included in miRNA profiling and integrated multivariate analysis due to unmatched miRNA measurements. Supporting changes can be found – page 8 (Design + Table 1), page 15 (ELISA; Figure 7 legend, lines ~484–490), and page 16 (flow cytometry figure includes ECOR63; Figure 8 legend, lines ~503–506).   Comments 13: Flow cytometry is performed only with EcN and ECOR12 (Lines 413–416). This prevents linking the ECOR63 cytokine phenotype to the maturation marker phenotype, thereby weakening strain-comparative conclusions. Response 13: Thank you. We addressed this directly by including ECOR63 in the extended phenotyping flow cytometry dataset so that ECOR63 cytokine tendencies can be discussed alongside maturation-marker phenotypes within the same phenotyping arm. This change can be found – page 16, Figure 8 (histograms include ECOR63) and legend, lines ~503–506.   Comments 14: miRNA assays are performed only with EcN and ECOR12 (Lines 425–428). Again, ECOR63 is absent—yet later the Discussion interprets ECOR63 as a distinct immunological “tone.” This should be either tested or removed/softened. Response 14: Thank you. We agree. We kept miRNA profiling restricted to the core integrated arm (control, EcN, ECOR12) and revised the Discussion/Conclusions to avoid over-claiming ECOR63 as a complete multi-omic “tone.” ECOR63 is now consistently framed as supporting context from extended phenotyping (ELISA/flow/OMV profiling), not as part of miRNA-driven integrated fingerprints. This change can be found – page 8 (Table 1 notes; miRNA core-only), page 11 (Statistics; integrated inference core-only) lines 399-409, and page 22, lines ~678–703 (Discussion paragraph explicitly distinguishing core vs extended and limiting polarization claims).   Comments 15: PCA integrates cytokines and miRNA and reports n = 60, but the shape legend includes only Control, EcN, and ECOR12 (Lines 456–457). PCA omits ECOR63 even though the cytokine ELISA includes it. This makes the multivariate “strain separation” claim incomplete; either include ECOR63 across all modalities, or explicitly state that PCA is only ECN vs ECOR12 vs control. Response 15: Thank you. We explicitly state that PCA/integrated multivariate analyses are restricted to the core integrated dataset (control, EcN, ECOR12) and clarify that ECOR63 is excluded from these analyses due to unavailable matched miRNA measurements. This clarification is present in the Methods/Statistics and in the figure caption/legend context for the PCA. This change can be found – page 11, lines ~392–409 (integrated analyses defined as core-only) and page 18, Figure 11 legend, lines ~545–549 (points correspond to donor-condition observations in the core dataset; ECOR63 not included).   Comments 16: The Discussion interprets strain-specific “tone” and explicitly contrasts ECOR12 vs ECOR63 (Lines 589–590). This is not fully supported because ECOR63 is not included in flow cytometry/miRNA experiments (Results lines 413–428). Either add ECOR63 to those assays or limit ECOR63 claims to cytokines/protein profiling only. Response 16: Thank you. We addressed this in two ways: (i) ECOR63 was added to flow cytometry within the extended phenotyping arm; and (ii) we revised the Discussion to ensure ECOR63 interpretations are limited to extended phenotyping outputs and are not presented as part of the integrated miRNA-based fingerprint. This change can be found – page 16, Figure 8 (flow includes ECOR63) lines 502-506 and page 22, lines ~678–703 (Discussion text limiting polarization/“tone” claims and explicitly positioning ECOR63 as contextual extended phenotyping).   Comments 17: The manuscript appropriately notes that DC polarization cannot be inferred from a single marker (Lines 602–605). Still, this argument should also motivate a more complete phenotype panel or functional readouts if claiming differential polarization. Response 17: Thank you. We agree and strengthened the Discussion by explicitly stating that the observed marker/cytokine/miRNA patterns support programming tendencies rather than definitive polarization, and that future work should expand marker panels and functional assays (e.g., HLA-DR, CD80/CD86, CCR7, T-cell priming/cytokine skewing). This change can be found – page 22, lines ~691–703 (highlighted paragraph specifying limits and proposing expanded phenotyping/functional readouts).   Comments 18: The Conclusions state broad strain-comparative claims; however, given that ECOR63 is not consistently included across assays and statistical frameworks, the conclusions should be tightened to match the actual datasets (cytokines vs flow/miRNA vs multivariate). • The manuscript itself summarizes that probiotic and commensal strains yield distinct fingerprints (Conclusions area, Lines 776–781). Rephrase conclusions to reflect what is supported across all platforms, and clearly separate “supported by full multi-omic panel” vs “supported only by ELISA/protein profiling”. Response 18: Thank you. We agree and revised the Conclusions to clearly separate: (i) findings supported by the core integrated dataset (control, EcN, ECOR12; cytokines + flow + miRNAs + integrated multivariate analyses), from (ii) findings supported by the extended phenotyping arm (adding ECOR63 for ELISA/flow/OMV profiling) and explicitly stating the limitation of missing matched miRNA for ECOR63 in integrated analyses. This change can be found – page 26, lines ~879–908 (Conclusions; highlighted), and reinforced in the “Taken together” synthesis – page 25, lines ~836–856. 4. Response to Comments on the Quality of English Language   Point 1: Frequent mid-word hyphenation / broken words (major readability problem) These appear throughout and should be removed in the clean manuscript (often caused by manual hyphenation or formatting export): Abstract: “candi-dates”, “den-dritic”, “monocyte-de-rived”, “matu-ration”, “strain-en-coded”, “mi-croRNAs” (Lines 12–37).  Methods: “fil-trate”, “swing-ing-bucket”, “ultracentri-fu-gation”, “cryogen-ic”, “phys-iologically” (Lines 283–287).  Results: “matu-ration”, “ex-posed”, “re-lease”, “evalu-ate” (Lines 395–401, 407–410, 426–428).  Discussion/late section: “inter-pretable”, “clar-ify”, “pat-tern”, “signifi-cance”, “ade-quacy”, “strat-egy”, “engi-neered” (Lines 721–752).    Suggestion: Ensure the editable source file (Word/LaTeX) has no manual hyphen characters inserted mid-word; re-export to PDF cleanly. Response 1: Thank you. We agree and removed manual mid-word hyphenation/broken words throughout the manuscript to improve readability and professionalism. This change is reflected across the revised document, including the Abstract and Methods/Results passages previously affected.   Point 2: Overlong, multi-clause sentences (reduce clarity; split into shorter sentences) Abstract contains several very long sentences with many clauses and statistics in the same line block (Lines 14–36).  Split into 4–5 shorter sentences: (background) → (what you did) → (key cytokine result) → (key miRNA result) → (statistics + conclusion). Response 2: Thank you. We agree and revised the Abstract by splitting long multi-clause sentences into shorter units following the recommended logic (background → approach → key cytokine finding → key miRNA finding → statistics/conclusion). This change can be found – page 1, lines 11–26.   Point 3: Consistency in spelling (choose one style and apply everywhere) The Abstract uses “TLR signalling” (British spelling) (Line 21) while elsewhere the manuscript uses “signaling” in the Methods section (Lines 334–336). Decide on American English (signaling) or British English (signalling) per journal preference and apply consistently.

Response 3: Thank you. We standardized spelling consistently across the manuscript (including “signaling/signalling”) to avoid mixed conventions and improve editorial consistency.

 

 

Point 4: Terminology consistency (avoid switching forms within the same passage)

In the Abstract, both “microRNAs (miRNAs)” and later “miRNAs” are used interchangeably; also, the keywords list shows “mi-croRNAs” due to formatting (Lines 14–30).  Define once (“microRNAs, miRNAs”), then use miRNA/miRNAs consistently.

Response 4: Thank you. We agree and standardized terminology by defining “microRNAs (miRNAs)” once and then using “miRNA/miRNAs” consistently throughout.

 

Point 5: Minor style/clarity polishing (tighten phrasing)

Examples where minor edits would improve clarity and flow:

• “DC maturation was evaluated using two complementary approaches…” is clear, but later wording becomes repetitive (“quantifications are shown…”, “results are shown…”) (Lines 394–417).

Suggestion: Vary structure and reduce “shown” repetition (e.g., “Figure X summarizes…”).

Response 5: Thank you. We edited phrasing in Results to reduce repetitive “shown” structures and used more varied figure-referencing language (e.g., “Figure X summarizes…”, “Figure X illustrates…”), improving flow and readability.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the major concerns raised in my previous report.