Per- and Polyfluoroalkyl Substance-Induced Skin Barrier Disruption and the Potential Role of Calcitriol in Atopic Dermatitis

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

-Major Comments

Comments 1: The paper positions calcitriol as a potentially protective modulator, but it remains unclear whether its observed effects are specific to PFAS-induced damage or reflect general anti-inflammatory and pro-differentiation roles in keratinocytes. The discussion could benefit from a clearer differentiation between these possibilities.

Response 1: We appreciate the reviewer’s insightful comment. To address this point, we have expanded the discussion section to acknowledge the possibility that the observed effects of calcitriol may not be limited to PFAS-induced damage. This addition aims to clarify the broader relevance of calcitriol’s effects and to address the specificity of the observed responses in our model. The revised text can be found in the Discussion section, page 13, lines 367-376.

Comments 2: While the suppression of VDR and CYP24A1 is demonstrated and contextualized with in silico findings, it would be valuable to more directly discuss whether PFAS exposure affects VDR nuclear translocation or ligand-binding in future directions or as limitations.

Response 2: We thank the reviewer for this thoughtful comment. In response, we have revised the discussion section to include the potential impact of PFAS exposure on VDR nuclear translocation and ligand binding as a mechanistic limitation that warrants further investigation. This addition aims to address the reviewer’s concern by acknowledging the current limitations and highlighting future directions for mechanistic validation. The revised text can be found in the Discussion section, page 13, lines 384 – page 14, lines 389.

Comments 3: The use of IC₂₅ values for PFAS treatments is appropriate. Still, a brief comment should be added in the discussion regarding the extrapolation of these concentrations to human-relevant exposure levels in real-world conditions.

Response 3: We appreciate the reviewer’s thoughtful comment. To address this point, we have added a brief statement in the Discussion section that acknowledges the limitations in extrapolating in vitro concentrations to real-world human exposure levels. Specifically, we now note that although the IC₂₅ concentrations used in this study are higher than typical PFAS levels observed in human serum, they were selected as practical experimental thresholds to induce early cellular responses without causing overt cytotoxicity. This addition clarifies the rationale for the selected concentration while addressing its relevance and limitations with respect to environmental exposure levels. The revised text can be found in the Discussion section, page 12, lines 313-316.

Comments 4: The stratification strategy (PFNA_H_A vs. PFNA_L_NA) is sound. However, since the atopic status and PFAS exposure are not fully orthogonal in design, a brief note on the potential for residual confounding (e.g., socioeconomic status, diet, genetics) would improve transparency.

Response 4: While our stratification strategy was designed to explore potential interactions, we agree with the reviewer that PFAS exposure and atopic status may not be completely orthogonal. In response, we have added a statement in the Discussion section acknowledging the possibility of residual confounding due to unmeasured variables such as socioeconomic status, dietary habits, or genetic background. While this limitation does not compromise the stratified analysis, it highlights the need for future population-based studies incorporating broader contextual and environmental factors. The revised text appears in the Discussion section (page 14, lines 390-396).

-Minor Comments

Comments 1: Consider adding one or two quantitative highlights (e.g., “533 PFNA-specific DEGs identified”) to increase impact and informativeness.

Response 1: We appreciate the reviewer’s suggestion. Quantitative highlights have already been presented in the Results section (page 6, lines 184-195) to inform the reader of key findings. However, we have ensured that the quantitative findings are appropriately emphasized where most relevant in the main text.

Comments 2: Slight rewording: “calcitriol may serve as a protective modulator” → “calcitriol demonstrates potential as a protective modulator.”

Response 2: We thank the reviewer for the helpful suggestion. We have revised the sentence accordingly to read “calcitriol demonstrates potential as a protective modulator,” which more clearly reflects the observed findings. The updated text appears on page 1, line 25.

Comments 3: Figure 3: Consider indicating time points (24 h, 48 h) more prominently in the panel titles.

Response 3: We thank the reviewer for the suggestion. To improve clarity, we have revised the figure by making the panel labels bold within the image. This adjustment helps distinguish each panel more clearly in relation to the gene names and the time point comparisons (24 h vs. 48 h).

Comments 4: Figure 6: Add clarification on whether CYP24A1 induction by calcitriol + PFAS is statistically different from PFAS or calcitriol alone.

Response 4: We appreciate the reviewer’s suggestion. To clarify this point in the main text, we revised the sentence to highlight the significance of the observed difference explicitly. The updated text appears on page 10, line 265-269. We believe this revised phrasing more clearly conveys the statistical difference between the groups.

Comments 5: While Student’s t-test is used, indicate whether normality assumptions were tested, especially for small group sizes in transcriptomic analyses.

Response 5: We thank the reviewer for this thoughtful comment. For our qPCR analyses, each condition included a minimum of three independent biological replicates, and each measurement was performed in technical triplicates to ensure accuracy and reproducibility. While formal normality testing was not feasible due to the limited number of biological replicates, we applied the Student’s t-test under the assumption of approximate normality, which is commonly accepted in qPCR-based gene expression studies with low technical variability. To improve the transparency of our statistical approach, we have clearly stated this in the Materials and Methods section.

Comments 6: Avoid phrases like “strong induction” or “robustly induced” unless accompanied by fold change or p-values.

Response 6: We appreciate the reviewer’s thoughtful suggestion. In response, we have revised expressions to more neutral and objective language in the main text. For example, the sentence:

“In contrast, calcitriol alone robustly induced the expression of CYP24A1, particularly at 48 h.”

has been revised to:

“In contrast, calcitriol alone led to a marked increase in CYP24A1 expression, particularly at 48 h.”

Similarly, in the sentence:

“Additionally, the strong induction of CYP24A1 under co-treatment conditions supports the notion of a synergistic interaction between PFAS and calcitriol,”

has been revised to:

“Additionally, the observed induction of CYP24A1 under co-treatment conditions supports the notion of a synergistic interaction between PFAS and calcitriol.”

We believe these revisions improve the objectivity and clarity of the manuscript while still accurately conveying the experimental findings.

Comments 7: Consider softening speculative statements (e.g., "suggesting PFAS create an inflammatory milieu...") with qualifiers like “may suggest” or “are consistent with.”

Response 7: We thank the reviewer for this helpful comment. In response, we have revised the speculative expression on page 10, line 277-279, to adopt a more cautious and scientifically appropriate tone.

Comments 8: Line 23: “PFASs contribute to AD-like skin pathology by impairing vitamin D receptor signaling and antimicrobial defense” could be more cautiously phrased as “PFASs may contribute…”.

Response 8: We appreciate the reviewer’s suggestion. In response, we have revised the statement on line 23 to adopt a more cautious and scientifically appropriate phrasing, in line with the reviewer’s recommendation.

Comments 9: Line 256: “gene expression was analyzed by RT-qPCR” – you might add that GAPDH was used as a reference gene here.

Response 9: We thank the reviewer for this helpful suggestion. In response, we have revised the sentence on page 4, line 142 and page 9, line 257 to specify that GAPDH was used as the reference gene in the RT-qPCR analysis.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

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

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Can be improved

Are all figures and tables clear and well-presented?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The manuscript 2.2 mentions “Notably, cells in the Combined group demonstrated the most pronounced morphological disruption, including marked loss of adhesion and increased heterogeneity in cell shape.” To support this argument, clearer cell morphology images and evidence of adhesion loss should be included.

Response 1: We thank the reviewer for the constructive comment. Upon review, we agree that the current images do not provide sufficient evidence to support the original statement. Therefore, we have removed the sentence describing morphological disruption in the Combined group to maintain scientific accuracy and clarity.

Comments 2: The introduction requires added examples linking PFAS to AD and analysis of their positive correlation mechanisms.

Response 2: We appreciate the reviewer’s thoughtful suggestion. While the original manuscript included several epidemiological studies linking PFAS exposure to atopic dermatitis, we agree that the mechanistic connections could be stated more clearly. Accordingly, we have added a sentence in the Introduction to briefly highlight potential mechanisms that may contribute to the observed associations. We thank the reviewer for helping us improve the clarity of this section. The revised text can be found in the Discussion section, page 2, lines 78-80.

Comments 3: The grammar and language usage should be improved and corrected.

Response 3: We thank the reviewer for this comment. The manuscript was previously reviewed by a professional English editing service Editage (https://www.editage.co.kr/) prior to submission. Nevertheless, in response to the reviewer’s suggestion, we have carefully re-checked the revised manuscript to ensure clarity and correctness in grammar and language usage. We appreciate the reviewer’s attention to detail. Should additional language improvements be required, we are willing to utilize the IJMS Rapid English Editing service to ensure clarity and accuracy.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: It would be enriching if, at the end of the discussion, the authors could address the therapeutic implications for patients with AD.

Response 1: We appreciate the reviewer’s thoughtful suggestion. While our study is based on in vitro findings, we agree that addressing potential therapeutic implications could enhance the translational relevance of the work. In response, we have added a concluding sentence at the end of the Discussion section (page 14, line 404-406) to cautiously suggest that targeting the VDR pathway with calcitriol may offer therapeutic value in managing AD symptoms, particularly those exacerbated by environmental factors such as PFAS exposure.