Review Reports - The Importance of Nutritional Aspects in the Assessment of Inflammation and Intestinal Barrier in Patients with Inflammatory Bowel Disease

Round 1

Reviewer 1 Report

The authors conducted an interesting and important study. In the opinion of the reviewer, the main limitation of this study is the very small sample size which might be responsible for the negative results. Further (and this relates to the small sample size) the authors did not differentiate between UC and Crohn’s disease. In the opinion of the reviewer, measuring mRNA tissue expression is not sufficient to predict barrier function.

Author Response

Response to Review Comments

We would like to thank the Editors and Reviewers for their comprehensive review. We have revised our paper in line with the suggestions and comments. Our responses are included below.

Reviewers' comments:

Reviewer#1:

1. The authors conducted an interesting and important study. In the opinion of the reviewer, the main limitation of this study is the very small sample size which might be responsible for the negative results. Further (and this relates to the small sample size) the authors did not differentiate between UC and Crohn’s disease. In the opinion of the reviewer, measuring mRNA tissue expression is not sufficient to predict barrier function.

Answer: Thank you for your comment. We agree that the main limitation of our study is the small number of participants in each study group, which may have affected results. We described this issue in the Discussion section. However, this is an initial study only, and we are planned further research in this area on a larger study group, with a separation of patients with Crohn's disease and ulcerative colitis.

In the present study, we conducted a preliminary study that allowed the evaluation of multiple parameters in various biological materials, including fecal organic acid profile, blood cytokines level and mRNA expression of selected parameters in intestinal tissue. However, we plan to conduct a study focusing on a comprehensive assessment of the intestinal barrier function, taking into account a wider group of parameters.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments for author File: Comments.docx

Author Response

Reviewer #2:

One of the conclusions that patients with active IBD are characterized by elevated levels of CRP and calprotectin, and reduced albumin, was already established in the field of IBD before this study.

Answer: Thank you for this comment. We agree with the Reviewer, however, we believe that it is important to present these data as they confirm correctly selected groups of patients (active vs inactive IBD). As is well known, the evaluation of disease activity according to the full Mayo criteria for UC and the Crohn's Disease Activity Index and Simple Endoscopic Score for Crohn Disease for CD, is not optimal and does not take biochemical parameters into account [1,2]. Therefore, it seems important to us to emphasize that patients in particular groups differed in the activity of commonly used parameters of inflammation.

[1] Freeman HJ. Use of the Crohn's disease activity index in clinical trials of biological agents. World J Gastroenterol. 2008 Jul 14;14(26):4127-30. doi: 10.3748/wjg.14.4127.

[2] Cooney RM, Warren BF, Altman DG, Abreu MT, Travis SP. Outcome measurement in clinical trials for Ulcerative Colitis: towards standardisation. Trials. 2007 Jun 25;8:17. doi: 10.1186/1745-6215-8-17.

In discussion, authors mentioned that lower levels of the SCFAs in patients with active IBD might be caused by the reduced consumption of fiber-rich foods and by the disruption of the gut microbiota reflected by reduced amounts of SCFA-producing bacteria. However, in general, fat is known as one factor to exacerbate IBD, and patients with IBD would try to reduce the intake of fatty acids, particularly when their disease are in active phase. In this study, the consumption of fatty acids was not considered, and the questionnaire focused on only fibers. We could not conclude that the reduction of the SCFA in stools was reflected by the reduced consumption of SCFA, which leads to the lower mRNA expression of tight junction proteins in the intestine, resulting in the exacerbation of IBD.

Answer: Thank you for your comment. It is true that we did not evaluate the consumption of fatty acids such us medium-chain fatty acids and long-chain fatty acids, which was due to the fact that they did not contribute to the production of SCFA or branched chain fatty acids. In addition, we excluded patients taking SCFA, which is available in supplement form. In our nutritional questionnaire, we focused on fiber-rich products, which are the main substrate for SCFA production in the human colon. On the other hand, the study has the disadvantage of not evaluating protein intake, which is marginally responsible for branched chain fatty acids production. We are currently working on another study in which we will improve the nutritional assessment, including the use of a 24-hour dietary interview.

We also agree with the Reviewer that, based on our study, we cannot identify the mechanism of lower mRNA expression of tight junction (TJ) proteins in active IBD. However, this was not the purpose of our study. In the study, we evaluated the relationship between TJ protein expression and disease activity. We observed that patients with active IBD had lower intestinal expression of occludin, claudin-2, and ZO-1 in intestinal tissue. This allowed us to make some hypotheses that need to be confirmed in further research, as we described in the discussion section.

Since the pathologies differ from ulcerative colitis and Crohn's disease, it would be necessary to check significant differences in each item between the two groups or to include patients with either UC or CD in this study.

Answer: Thank you for your comment. We agree with the Reviewer. In the discussion, we described that one drawback of the study was the small group of patients (line 416), which prevented us from separating patients into UC and CD. We also highlighted that we are aware that the lack of isolation of these patients may be the reason for such findings (line 383). However, this is an initial study only, and we are planned further research in this area on a larger study group, with a separation of patients with Crohn's disease and ulcerative colitis.

There is a mistake in the hemoglobin value for active IBD in Table 1.

Answer: Thank you for your comment. We have made the correction.

Since Tables 3-5 have the same structure, it would be easier to understand for readers if they were combined into one.

Answer: Thank you for your suggestion. We have revised the tables for better clarity.

Succinate and Succinic acid are mixed in the text and should be unified.

Answer: Thank you for your suggestion. Both forms (Succinate and Succinic acid) are deliberately used. In the lumen of the gastrointestinal tract, organic acids occur as anions i.e. acetate, propionate, succinate. Some researchers we cited in the manuscript measured succinate levels. We presented the result for the concentration of succinic acid.

Within the Table, align the significant digits.

Answer: Thank you for your suggestion. We have made the correction as suggested.

UC and CD have disease-specific indexes for activities. They should be adopted for the assessments of the disease activities in this study.

Answer: Thank you for your comment. Patients were divided into groups with inactive and active disease according to the full Mayo criteria for UC and the Crohn's Disease Activity Index and Simple Endoscopic Score for Crohn Disease for CD as we described in paragraph “2.1. Study Population” (line 94-98).

Author Response File: Author Response.docx

Reviewer 3 Report

1.Summary

The manuscript aimed to characterize the associations between nutritional, inflammatory and intestinal barrier parameters in patients with inflammatory bowel disease (IBD). To do this, they comprehensively assessed several important parameters including nutritional status, fecal short-chain fatty acid profile, cytokine levels in serum, mRNA expression of enzymes and tight junction proteins obtained from active and inactive IBD and controls. Overall, the experiments were well designed, and all the measurements were conducted by following well established standard. The methods are sufficiently described, and the results are well presented. Tables and figures are organized in a way that is easy to understand. The results are well interpreted and discussed, and the study highlights the need for a holistic approach to manage IBD patients with concomitant personalized nutritional treatment to achieve clinical remission and mucosal healing. The following are a few major and minor points that the authors need to address before acceptance.

2. Major point

A big issue for the experimental design is that there is a gender bias for the studied population. In total there are 35 patients and 65% (23/35) are male. Specifically, 78% and 67% patients are male in inactive and activate IBD groups, respectively. The authors need to justify in discussion section whether this will influence the generality of the conclusion obtained (e.g. whether the conclusion only holds true for male population and does not apply to female population). To address this question, the authors can try to repeat all the analyses, but further separating each group into male and female population. This may be problematic for inactive IBD and control groups because of small sample size after separation, but this analysis should be possible for the active IBD group.

3. Minor points

Page 5, Table 1, row 2 and column 2: the number is not center aligned.

Page 6, line 228: active IBD group is firstly described (Table 4), followed by inactive IBD group (Table 3). But the order of tables is inconsistent with the order of their descriptions. A suggestion is to switch Table 3 with Table 4.

Page 7, Table 6: n=10 and 5 for inactive IBD and controls groups, respectively. One patient data is missing for each group. The authors need to add a table note to denote why there are missing patients/data.

Page 8, Table 7: the same issue as mentioned above. There are missing patients/data for each group. The authors need to add a table note to denote why there are missing patients/data.

Author Response

Reviewer #3:

A big issue for the experimental design is that there is a gender bias for the studied population. In total there are 35 patients and 65% (23/35) are male. Specifically, 78% and 67% patients are male in inactive and activate IBD groups, respectively. The authors need to justify in discussion section whether this will influence the generality of the conclusion obtained (e.g. whether the conclusion only holds true for male population and does not apply to female population). To address this question, the authors can try to repeat all the analyses, but further separating each group into male and female population. This may be problematic for inactive IBD and control groups because of small sample size after separation, but this analysis should be possible for the active IBD group.

Answer:

Thank you for your comment. We agree that one of the drawbacks of the study is the preponderance of men among participants. However, this is only a preliminary study. We are planning further research on a larger research group, which will allow us to avoid this limitation. At the same time, we conducted a statistic analysis as suggested by the Reviewer. Based on it, we added short paragraph to the text (Results section):

“3.6. Investigation of possible gender bias for the studied population

In the case of the most numerous group of subjects (active IBD), the influence of gender on the values of the parameters tested was checked. With the exception of BMI (medians: 19.2 kg/m2 vs. 23.6 kg/m2, p=0.032, for females and males, respectively), no gender effect was found for all other parameters (p within the range 0.1247 – 1.000).”

Page 5, Table 1, row 2 and column 2: the number is not center aligned.

Answer: Thank you for your suggestion. We have made the correction as suggested.

Page 6, line 228: active IBD group is firstly described (Table 4), followed by inactive IBD group (Table 3). But the order of tables is inconsistent with the order of their descriptions. A suggestion is to switch Table 3 with Table 4.

Answer: Thank you for your comment. We have changed the order of the Tables as suggested.

Page 7, Table 6: n=10 and 5 for inactive IBD and controls groups, respectively. One patient data is missing for each group. The authors need to add a table note to denote why there are missing patients/data.

Page 8, Table 7: the same issue as mentioned above. There are missing patients/data for each group. The authors need to add a table note to denote why there are missing patients/data.

Answer: Thank you for your suggestions. Due to insufficient biological material, the SCFA profile in the stool was not determined in 2 patients (one patient with inactive IBD and one control) and the concentration of cytokines in the blood in one patient in each group. The description of Table 6 and 7 has been modified.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Figure 1: The authors should provide (as a separate supplementary figure) detailed information regarding the different targets shown in Figure 1 by dividing data into UC and CD.

The authors must include a separate paragraph where they explicitly list all limitations of the study (small sample size, intestinal barrier marker etc.)

“Data not shown”: Please add supplementary figures for every statement “data not shown”

Author Response

Response to Review Comments

We would like to thank the Editors and Reviewers for their comprehensive review. We have revised our paper in line with the suggestions and comments. Our responses are included below.

Reviewers' comments:

Reviewer#1:

1) Figure 1: The authors should provide (as a separate supplementary figure) detailed information regarding the different targets shown in Figure 1 by dividing data into UC and CD.

Answer: Thank you for your suggestions. We also believe it would enrich our work. However, since the number of participants in each subgroup is too small, it is not statistically possible to make such a comparison. We are conducting further studies in this area on a larger group of participants, in which we want to make such a division (UC vs CD), also taking into account disease activity (inactive UC vs active UC; inactive CD vs active CD).

2) The authors must include a separate paragraph where they explicitly list all limitations of the study (small sample size, intestinal barrier marker etc.)

Answer: Thank you for your suggestions. We have added an additional paragraph about the main limitations of the study as suggested (line 428-436).

“The main limitations of our study are the small number of participants in each study group and the incomplete dietary assessment. SCFAs are mainly formed from dietary fiber, especially the soluble fractions. However, some SCFAs and branched fatty acids, such as isovalerate and isobutyrate, can be formed by bacterial fermentation of amino acids in the large intestine. Hence, it is necessary to conduct a complete dietary assessment, including a 24-hour dietary interview. Due to the preliminary nature of the present study, we made a limited evaluation of the intestinal barrier markers. Therefore, we are conducting further research in this area on a larger group of participants, including a broader assessment of intestinal barrier parameters.”

3) “Data not shown”: Please add supplementary figures for every statement “data not shown”

Answer: Thank you for your comments. We have enclosed a supplement containing figures as suggested.

Author Response File: Author Response.pdf

Reviewer 2 Report

Thank you for responding to the review. The sentences for the exclusion of the patients taking SCFA, and the limitation of the questionnaire without protein consumption should be added to the article. Also, references to the fact that the consumption of fatty acids did not contribute to the production of SCFA or branched chain fatty acids might help readers to understand this study.

Author Response

Response to Review Comments

We would like to thank the Editors and Reviewers for their comprehensive review. We have revised our paper in line with the suggestions and comments. Our responses are included below.

Reviewers' comments:

Reviewer#2:

Answer:

Thank you for your comment. We clarified the information that patients taking SCFA-containing supplements were excluded from the study (line 109-110). We have also added an additional paragraph about the main limitations of the study, including information about the shortcomings of our nutrition questionnaire in Discussion section (line 428-436).

Author Response File: Author Response.pdf