Review Reports - A Hot-Spring Water Improves Inflammatory Conditions in an Injury-Induced Atopic Dermatitis Mouse Model by Regulating Skin Barrier Function

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary:

This research article investigated the effects of Arifuku hot-spring water on management of atopic dermatitis. Several parameters were observed on the effect of hot-spring water on atopic dermatitis: skin barrier function, inflammation, filaggrin, TRPV4 level in the induced mouse model. Hairless mice was used in the study. The hairless mice were subjected to tape-stripping to create the atopic dermatitis-alike barrier injury. Then the mice were treated with hotspring water (treatment group) or tap water (control group). The parameters such as TEWL, skin histology, fliaggrin, TRPV4, CD8+, T cells and IL-4. In this study, it was discovered that hot-spring water can fasten the recovery of TEWL and maintained high water content by day 3. For treatment group, mice which were treated with hot-spring water showed thinner stratum spinosum, well-formed stratum granulosom, and early reappearance of stratum corneum. This is a sign of the skin barrier recovery. The expression of filaggrin improved significantly in the upper epidermis of the treatment group, but TRPV4 had no change. Significant anti-inflammatory activity was exhibited in hot-spring water, as indicated in the reduction of CD8+ T cells and IL-4 expression. In the discussion, the authors suggested that due to the presence of minerals, such as sodium, calcium, magnesium, bicarbonate, chloride, sulfate, and strontium, they could exhibit anti-inflammatory activity and increase keratinocyte proliferation and maturation. As a conclusion, this study had proven that Arifuku hot-spring water is effective in repairing the skin barrier, reducing the skin inflammation in atopic dermatitis-like mouse model.

Strengths:

This study explores the management of atopic dermatitis using natural and safe alternatives. Authors had used an approved mouse model that mimics the effect of skin thinning by using tape-stripping method.
The study was compared between hot-spring water and tap water (negative control) using standard and clear experimental design, and the effects were evaluated using immunostaining, measurement of TEWL, filaggrin, and relevant immune biomarkers to support the conclusion.
The mineral composition in the hot-spring water was analysed, and the presence of minerals was correlated with the effects in recovery of skin barrier.

Comments:

The treatment protocol was not clear. Authors must improve this.
Why the data is only limited to 3 days post-injury?
Authors need to explain how did the presence of minerals/specific minerals helped in the anti-inflammatory activity or modulating the keratinocytes?
TRPV4 is evaluated, but its importance in skin recovery was not properly discussed.
How did the mice receive the treatment (hot-spring water/tap water)? Any specific volume?
How many mice were allocated per group?
Did this experiment receive animal ethics approval?

Author Response

Strengths:

This study explores the management of atopic dermatitis using natural and safe alternatives. Authors had used an approved mouse model that mimics the effect of skin thinning by using tape-stripping method.

The study was compared between hot-spring water and tap water (negative control) using standard and clear experimental design, and the effects were evaluated using immunostaining, measurement of TEWL, filaggrin, and relevant immune biomarkers to support the conclusion.

The mineral composition in the hot-spring water was analysed, and the presence of minerals was correlated with the effects in recovery of skin barrier.

Comments:

The treatment protocol was not clear. Authors must improve this.

Response: According to the reviewer’s suggestion, the treatment protocol is clearly stated in the revised manuscript.

Why the data is only limited to 3 days post-injury?

Response: In our initial experiments, we found that the tape-stripping injury had significantly improved by 7 days after tape-stripping, even in the tap water-treated mice. As a result, there was no significant difference between the tap water-treated and hot spring water-treated mice in terms of skin pathology at the tape-stripped areas. Hence, to avoid confusion, we conducted our detailed investigation up to 3 days of treatment, where the difference was maximal. Since this point was raised by the reviewer, we have included this information in the revised manuscript.

Authors need to explain how did the presence of minerals/specific minerals helped in the anti-inflammatory activity or modulating the keratinocytes?

Response: According to the reviewer’s suggestion, we have discussed the possible role of minerals present in hot-spring water in improving skin condition in the injury-induced atopic dermatitis mouse model. Previous studies have investigated the role of strontium in controlling inflammation at the molecular level, as well as its involvement in keratinocyte maturation and filaggrin expression. The hot-spring water contained a high concentration of strontium. In the revised manuscript, we have discussed how elevated strontium levels could influence the pathology of the injury-induced atopic dermatitis model.

TRPV4 is evaluated, but its importance in skin recovery was not properly discussed.

Response: According to the reviewer’s suggestion, we have revised this section of the discussion to include the role of TRPV4 in atopic dermatitis and to relate our findings to its known functions. Since TRPV4 expression did not differ between tap water-treated and hot-spring water-treated tape-stripped skin, we suggest that TRPV4 may not play a major role in the beneficial effects of hot-spring water observed in this injury-induced atopic dermatitis mouse model.

How did the mice receive the treatment (hot-spring water/tap water)? Any specific volume?

Response: According to the reviewer’s suggestion, we have described how the mice receive treatment in the Materials and Methods section.

How many mice were allocated per group?

Response. In this study, five mice were allocated to each treatment group at each time point. This information is mentioned in the Materials and Methods section of the revised manuscript.

Did this experiment receive animal ethics approval?

Response: Our study was approved by the Ethical committee of the Shimane University School of Medicine. The Approval number is IZ5-35. This information was given in the Materials and Methods section of revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

I found your article interesting and impressive. Please embedded in the context see my comments and suggestions.

Comments for author File: Comments.pdf

Author Response

Please find the responses to the Reviewer's comments in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this report by Nagai and colleagues, the authors evaluate the protective effect of hot spring water in a tape stripping mediated atopic dermatitis (AD) model in vivo. By composition analysis, the hot spring water (HSW) was shown to be enriched with minerals and trace elements. Topical application of HSW rescued trans epidermal water loss (TEWL) associated with tape stripping compared to tap water. Histological analysis of murine skin sections also revealed better stratum spinosum and granulosum layers with reduced epidermal thickness. However, transient receptor potential vanilloid 4 (TRPV4), a protein associated with better barrier function did not show any significant change. Notably, filaggrin (FLG), a natural moisturizer protein is reduced in both tape stripped and non-tape stripped adjacent skin while HSW treatment significantly rescued FLG levels in the latter. Intra-epidermal/dermal infiltration of CD8 T cells and IL-4, a key cytokine involved in inflammatory response were shown to be elevated in tape stripped skin which were restored to normal levels upon HSW treatment. Overall, this study demonstrates the therapeutic benefit of HSW in a murine model of AD. Though this study is interesting, there are several lacunae, data discrepancy and lack of critical controls which need to be addressed prior to publication.

Comments:

Though authors provide data on the mineral composition of hot spring water, this could have also been done for the tap water that has been used in this study as a control, so that a side by side comparison of the composition would have possibly pointed out the key ingredient/s in hot spring water that could be responsible for the observed effect
There is some discrepancy in the thickness of epidermis seen in Figure 2e and f and Figure 3d and e. Despite both figures represent skin section histology from the same group of mice (tap water and hot spring water treated mice) at day 3 but there is tremendous difference in the thickness of epidermis between these figures.
The interpretation of results from both Figure 2 and 3 are merely qualitative and no quantitative data is shown. At least authors could measure the relative depth of the distinct layers of the skin (supranasal layers or stratum spinosum) in multiple histological images from different mice and should provide a quantitative bar graph.
In line now 296, authors state that Figure 5b and 5c shows the data for FLG IHC on day 2 but in figure legends it is mentioned as day 1. Which is correct?
Further, there is lack of methodological details on how much water was applied and the duration of the application. Critically, the differential effect of hot spring water on FLG expression in tape stripped and non-tape stripped areas calls for provision of accurate details to be provided on whether water was applied only to tape stripped areas or it extended to non-tape stripped areas as well. How much of the surface area of the mice skin was treated? Was there any specific quantity ratio (water ml/cm2 area) used for the

application of water? These details are missing.

For Figure 7, authors need to provide the DAPI counterstaining data . This will indicate if the CD8 and Il-4 positive cells are located within the intraepidermal or intra dermal areas.

Author Response

Comments:

Though authors provide data on the mineral composition of hot spring water, this could have also been done for the tap water that has been used in this study as a control, so that a side by side comparison of the composition would have possibly pointed out the key ingredient/s in hot spring water that could be responsible for the observed effect

Response: We thank the reviewer for this valuable comment. Unfortunately, the measurement of the mineral content of the tap water was not included in our study design; therefore, we are unable to provide specific data on the mineral composition or pH of the tap water used. However, previous studies have reported that the mineral content of Japanese tap water is generally low, and its hardness is classified as soft. Based on this, we consider that the relatively high mineral concentration in hot-spring water may contribute to its beneficial effects on the pathology of injury-induced atopic dermatitis. As this is an important point, we have included a discussion of this aspect in the revised manuscript.

There is some discrepancy in the thickness of epidermis seen in Figure 2e and f and Figure 3d and e. Despite both figures represent skin section histology from the same group of mice (tap water and hot spring water treated mice) at day 3 but there is tremendous difference in the thickness of epidermis between these figures.

Response: To establish the injury-induced atopic dermatitis mouse model, the left dorsal skin was subjected to tape stripping, while the right dorsal skin was left intact. The photomicrographs in Figure 2 show the histology of the tape-stripped left dorsal skin in both tap water-treated and hot-spring water-treated mice. In contrast, Figure 3 presents the histology of the non–tape-stripped right dorsal skin from the same mice. We examined the right dorsal skin to determine whether tape stripping on one side of the dorsum induced any reactive changes on the opposite, non–tape-stripped side. Some reactive changes were observed in the non–tape-stripped right dorsal skin; however, these alterations were less pronounced than those in the tape-stripped left dorsal skin. Consequently, the epidermal thickness in Figure 3 appears much lower than that shown in Figure 2. We acknowledge that our initial presentation of these data may have caused confusion. In response to the reviewer’s comment, we have revised the figure descriptions and corresponding text in the manuscript to clarify this point.

The interpretation of results from both Figure 2 and 3 are merely qualitative and no quantitative data is shown. At least authors could measure the relative depth of the distinct layers of the skin (supranasal layers or stratum spinosum) in multiple histological images from different mice and should provide a quantitative bar graph.

Response: We thank the reviewer for raising this point. In accordance with the reviewer’s suggestion, the thickness of the stratum spinosum was measured and included in the revised manuscript. The results showed a significant decrease in stratum spinosum thickness in the tape-stripped areas of the dorsal skin of mice treated with hot spring water. These data have been incorporated into the revised manuscript.

In line now 296, authors state that Figure 5b and 5c shows the data for FLG IHC on day 2 but in figure legends it is mentioned as day 1. Which is correct?

Response: We apologize for the typographical errors. Filaggrin immunostaining was performed using skin samples from mice on days 0, 2, and 3 after tape stripping. The mistake has been corrected in the revised manuscript.

Further, there is lack of methodological details on how much water was applied and the duration of the application. Critically, the differential effect of hot spring water on FLG expression in tape stripped and non-tape stripped areas calls for provision of accurate details to be provided on whether water was applied only to tape stripped areas or it extended to non-tape stripped areas as well. How much of the surface area of the mice skin was treated? Was there any specific quantity ratio (water ml/cm2 area) used for the application of water? These details are missing.

Response: According to the reviewer’s suggestion, the method section was amended, and how the water was applied was stated clearly.

For Figure 7, authors need to provide the DAPI counterstaining data . This will indicate if the CD8 and Il-4 positive cells are located within the intraepidermal or intra dermal areas.

Response: We appreciate the reviewer’s comment. Unfortunately, during the immunofluorescence staining for CD8 and IL-4, the tissue was not counterstained with Hoechst. Therefore, nuclear counterstaining data could not be included in the revised manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript contains results of the mouse model research important form the point of view of factors contributing to development and perpetuation of clinical symptoms of atopic dermatitis, causing considerable lowering of everyday life quality in affected subjects.

The introduction comprehensively presents the topic and brings the overview of hitherto performed studies regarding pathomechanisms that have been addressed by the authors, thus justifying their tackling on this issue in the current project. I think this section is a little bit too expanded: the paragraph contained in lines 85 through 96 could easily be omitted. Moreover, I cannot fully agree with the statement, that “While most therapies aim to control the immune response, there is relatively little emphasis given on improving the natural barrier function of skin”. Emollients and proper skin hydration has been mentioned in current guidelines for AD management and treatment as basic on every stage of the disease, therefore, we cannot say it is given little recognition or neglected. The authors may consider rephrasing this fragment.

Description of methods is in my opinion sufficient and provides all necessary data to guide the experiments aiming at results’ reproducibility. What deserves to be underlined, the authors have highlighted and indicated limitations of the study as a whole and of the employed model, which does not contain all features of allergic atopic inflammation.

I would like to highlight some of the possible clinical aspects of the study results, which may be contained in the manuscript at authors’ discretion. Namely, since the results have been collected up to day 3, how would the results be possibly extrapolated onto longer periods of the duration of the disease? This might be of importance in view of the chronic, sometimes relapsing-remitting course of AD.

Another aspect which I’d like to bring into attention is associated purely with the hot spring water used in the study. Has this been commercialized as an accessory product for AD management? Are the authors aware of the available similar products in different areas of the world? And finally, can this be considered a perspective for future modifications of AD management guidelines? It may be interesting to read some highlights and/or speculations regarding these aspects of AD management.

Author Response

Response: We also agree that the paragraph may not be essential. Therefore, in accordance with the reviewer’s suggestion, we have re=written this section of the manuscript.

Moreover, I cannot fully agree with the statement, that “While most therapies aim to control the immune response, there is relatively little emphasis given on improving the natural barrier function of skin”. Emollients and proper skin hydration has been mentioned in current guidelines for AD management and treatment as basic on every stage of the disease, therefore, we cannot say it is given little recognition or neglected. The authors may consider rephrasing this fragment.

Response: We agree with the reviewer. We have amended this section of the introduction section.

Response: We thank the reviewer for the comment.

Response: As a preliminary experiment, we examined the tape-stripping model and the potential effects of hot-spring water on AD pathology over an extended time period. We found that after 7 days, the skin injury was significantly improved even in tap water–treated mice. However, on day 3, the difference between the tap water and hot-spring water groups was significant. Therefore, we limited our study to the 3-day time point. This constitutes a limitation of our tape-stripping AD model, which is discussed in the revised manuscript.

Another aspect which I’d like to bring into attention is associated purely with the hot spring water used in the study. Has this been commercialized as an accessory product for AD management?

Response: The hot spring water has not been commercialized.

Are the authors aware of the available similar products in different areas of the world?

Response: To our knowledge, hot-spring water has not been commercialized as an accessory product for AD management.

And finally, can this be considered a perspective for future modifications of AD management guidelines? It may be interesting to read some highlights and/or speculations regarding these aspects of AD management.

Response: We believe that hot-spring water has potential as an adjunctive approach for managing atopic dermatitis. However, further studies are needed to clarify its therapeutic mechanisms and evaluate its efficacy in combination with standard treatments. Only then can its inclusion in AD management guidelines be appropriately considered.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have addressed my comments