Preventative Effects of Milk Fat Globule Membrane Ingredients on DSS-Induced Mucosal Injury in Intestinal Epithelial Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors

the paper is well written and organized, with interesting results concerning the effect of MFGM components on mucin production in intestinal cells.

I have some comments and questions regarding the following points:

Introduction: since MFGM are naturally present in breastmilk but not in infant formulas, some information on the current use of MFGM in infant formulas, and which infant populations could benefit from it, should be mentioned.

Figure 2A: are the p-values 5% and 10%MPL correct (is 10% MPL really lower than 5%)?

Figures 2B and 2D: the data related to the negative control and 10% MFGM (fig 2B), as well as those related to the positive control DSS+blank digesta (fig 2D) are very dispersed.

Figure 2C: despite 5% MPL+DSS shows the same trend of 2 and 10% treatments, it appears as not significant, probably as a result of data dispersion. 

With such high dispersion of data, especially in the case of positive and negative control, it is hard to tell if treatments produce a significant effect or not.  How could you explain this variability? I suggest to revise the experimental conditions and results of TEER and FD4 permeability results.

Lines 347-348: the sentence "cells were stained with PAS and Alcian blue to indicate the presence of acidic/sialylated and neutral mucins, respectively" is to be revised, since the activity of the mentioned dyes is the opposite.

Line 419: I would remove from "and MPL" to the end of the sentence.

Discussion: please expand by adding some references about the potential application of MFGM and its components to reduce intestinal damage.

Best regards

Author Response

Reviewer 1:

Dear authors

the paper is well written and organized, with interesting results concerning the effect of MFGM components on mucin production in intestinal cells.

I have some comments and questions regarding the following points:

Introduction: since MFGM are naturally present in breastmilk but not in infant formulas, some information on the current use of MFGM in infant formulas, and which infant populations could benefit from it, should be mentioned.

AU: Thank you for your suggestion. We’ve included your suggestion in line 85.

Figure 2A: are the p-values 5% and 10%MPL correct (is 10% MPL really lower than 5%)? Figures 2B and 2D: the data related to the negative control and 10% MFGM (fig 2B), as well as those related to the positive control DSS+blank digesta (fig 2D) are very dispersed. Figure 2C: despite 5% MPL+DSS shows the same trend of 2 and 10% treatments, it appears as not significant, probably as a result of data dispersion. With such high dispersion of data, especially in the case of positive and negative control, it is hard to tell if treatments produce a significant effect or not.  How could you explain this variability? I suggest to revise the experimental conditions and results of TEER and FD4 permeability results.

AU: Thank you for your comments. We respectfully prefer to leave the experimental conditions and results of the TEER and FD4 permeability results as shown as the data presented accurately represents our observations, including those of data dispersion. It would not be surprising to us that the data dispersion may be inherent, in part, to the complexity of the ingredients being studied.

Lines 347-348: the sentence "cells were stained with PAS and Alcian blue to indicate the presence of acidic/sialylated and neutral mucins, respectively" is to be revised, since the activity of the mentioned dyes is the opposite.

AU: We appreciate the attention to detail. We have revised the sentence.

Line 419: I would remove from "and MPL" to the end of the sentence.

AU: We revised the sentence for clarity.

Discussion: please expand by adding some references about the potential application of MFGM and its components to reduce intestinal damage.

AU: We have added lines 489-497.

Best regards

 

Reviewer 2 Report

Comments and Suggestions for Authors

I had the pleasure of reading your paper on MFGM ingredients effects on intestinal CaCo cells with DSS-induced injury. The effects on Mucin depletion is quite evident from TEER measurements. I suggest to clarify the results of the bioinformatic effort of the prediction model created beacuse it is not completely clear. Another suggestion is to add a potential ex vivo experiment to further implement the findings (merely descriptive), I suggest intestinal organoids. 

Comments on the Quality of English Language

The english is simple and fluent. Minor editing needed

Author Response

Reviewer 2:

I had the pleasure of reading your paper on MFGM ingredients effects on intestinal CaCo cells with DSS-induced injury. The effects on Mucin depletion is quite evident from TEER measurements. I suggest to clarify the results of the bioinformatic effort of the prediction model created beacuse it is not completely clear. Another suggestion is to add a potential ex vivo experiment to further implement the findings (merely descriptive), I suggest intestinal organoids. 

 

AU: Thank you for your suggestions! We have revised section 3.4 regarding the bioinformatics and added a few sentences about intestinal organoids for future research in lines 534-538.

 

 

Reviewer 3:

 

The Authors reporte the results of their experimental in vitro study on “Preventative effects of milk fat globule membrane ingredients on DSS-induced mucosal injury in intestinal epithelial cells”.

 

The topic is especially interesting as stated by Authors at 40-45 lines of Introduction section “…Perturbations in the mucosal barrier through various stimuli such as pathogenic infection, dysbiosis of the gut microbiota, various medications, stress, and poor nutrition increases the host’s susceptibility to damage and potentiates the onset of intestinal barrier dysfunction-related diseases, such as inflammatory bowel disease (IBD) and colitis [4]. Furthermore, mucosal barrier dysfunction predisposes individuals to development of food allergies, celiac’s disease, and diabetes..”

 

The study was well designed and methodologically executed. It was completed by the statistical analysis.

 

AU: Thank you!

 

In Section Conclusions The statement "....The findings from this work suggest that the MFGM can be used as a preventative nutritional component to reduce risk of intestinal barrier damage through mucin depletion.." in my opinion is inappropriate.

 

Being an experimental in vitro study, the Authors could add the limitations of the study in the conclusions section hoping to propose an in vivo study.

 

AU: Thank you for the suggestion. We understand your point and have revised the conclusions section with the following sentence “The findings from this work warrant further exploration of the MFGM to reduce intestinal barrier damage using more complex models such as intestinal organoids that more closely mimic the heterogeneity of the intestinal epithelium and in vivo models, as a major limitation of this study is the lack of interaction with the gut microbiota” in lines 534-538.

 

Reviewer 3 Report

Comments and Suggestions for Authors

please see attachment.

Comments for author File: Comments.pdf

Author Response

Thank you for your review.

Reviewer 4 Report

Comments and Suggestions for Authors

The paper by Kosmerl et al describes the effect of MGFM on the state of intestinal development and describes its beneficial effects on the state of integrity.

The introduction is very rich in bibliographical references and well described. The authors have included a lot of information and have clearly clarified the current state of the literature.

The methods are described well and with particular attention to all methodological details.

Minor revisions:

the changes induced by MFGM treatment on TEER should be related to the state of ion channels. Membrane current evaluation experiments should be added to validate the effect on TEER and to identify a possible player for the system.

The conclusion of line 269 < suggesting fortification of the mucosal barrier> should be supported with IF experiments to verify the integrity of the barrier.

The graphs in figure 2 are not uniform with each other, neither in size nor in style.

In line 304 the authors write:< With additional evidence, these findings may suggest an anti-cancer role of 304 the MFGM and its components as previously proposed [49,50].>. The references reported alone are not sufficient to draw such a conclusion without any experimental evidence in the model proposed by the authors. Experiments should be performed to evaluate the effect on some tumor biomarkers.

 

The graphs of figures 2 and 3 (in which only the relationships between the different treatments are shown) require at least some IF images to confirm the data on the alteration or otherwise of the barrier.

In figure 4 the scale bars are missing both in the images and in the legend.

Finally, the discussion is well structured and well argued.

Correct numerous typos and style errors

Comments on the Quality of English Language

Correct numerous typos and style errors

Author Response

Reviewer 4:

The paper by Kosmerl et al describes the effect of MGFM on the state of intestinal development and describes its beneficial effects on the state of integrity.

The introduction is very rich in bibliographical references and well described. The authors have included a lot of information and have clearly clarified the current state of the literature.

The methods are described well and with particular attention to all methodological details.

AU: Thank you!

Minor revisions:

the changes induced by MFGM treatment on TEER should be related to the state of ion channels. Membrane current evaluation experiments should be added to validate the effect on TEER and to identify a possible player for the system.

AU: Thank you for this observation and suggestion. On the issue of elucidating the relationship of our TEER measurements and its relationship to ion channels, we have to mention that our main objective in this work is to measure and describe the effects of MFGM on the cellular mucin production. We agree that TEER can be related to ion channels; however, this is a subject of future research in our group, and for the present work, we must accept just a description of the effect on the cell, and its implication with effect on tight junction among them.

 

The conclusion of line 269 < suggesting fortification of the mucosal barrier> should be supported with IF experiments to verify the integrity of the barrier.

AU: For clarification, we have revised this sentence to say “mucus layer” rather than “mucosal barrier.”

The graphs in figure 2 are not uniform with each other, neither in size nor in style.

AU: We have updated the figures for uniformity in both style and size. Each graph frame is set to 3x2 and we double checked that all font sizes are the same.

In line 304 the authors write:< With additional evidence, these findings may suggest an anti-cancer role of 304 the MFGM and its components as previously proposed [49,50].>. The references reported alone are not sufficient to draw such a conclusion without any experimental evidence in the model proposed by the authors. Experiments should be performed to evaluate the effect on some tumor biomarkers.

AU: We have deleted the sentence.

The graphs of figures 2 and 3 (in which only the relationships between the different treatments are shown) require at least some IF images to confirm the data on the alteration or otherwise of the barrier.

AU: We selected the use of a variety of approaches (TEER/FD4 permeability, mucin gene expression, as well SEM and mucin staining) to illustrate our findings that MFGM/MPL prevents DSS-induced mucin depletion as our primary focus was on mucin production. We are currently unaware of any IF techniques for mucin specifically, but the effects of MFGM ingredients on tight junctions, for example, using IF has been previously disclosed in Jiang et al (2022). https://pubmed.ncbi.nlm.nih.gov/35716863/ We mentioned these findings in line 264.

In figure 4 the scale bars are missing both in the images and in the legend.

AU: The scale bars and magnification are shown in the bottom right and left, respectively, in the images.

Finally, the discussion is well structured and well argued.

AU: Much appreciated, thank you for the feedback!

Correct numerous typos and style errors

AU: We have made corrections for these errors. Thanks!