Weissella koreensis KJ, Which Increases Gut Tight Junction Protein Expression, Alleviates TNBS-Induced Colitis by Suppressing Inflammatory Cytokines

Round 1

Reviewer 1 Report

This manuscript, authored by Kim et al., delves into the anti-inflammatory properties of Weissella koreensis KJ, isolated from the traditional Korean dish Kimchi. The researchers isolated W. koreensis KJ and investigated its impact on TNBS-induced colitis through various biochemical assays and microscopic analyses. Initially, they examined the expression of pro-inflammatory cytokines in macrophages following LPS treatment, comparing control cells with those treated with W. koreensis KJ. Subsequently, they evaluated its efficacy in a mouse model of TNBS-induced colitis, observing recovery in weight loss, colon inflammation, and shortening upon administration of W. koreensis KJ. Notably, the study revealed a reduction in the expression of pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 following oral administration of W. koreensis KJ. Finally, the authors demonstrated that W. koreensis KJ rescued the expression of gut tight junction proteins compared to the TNBS-treated control group. In summary, this research provides valuable insights into the anti-inflammatory effects of W. koreensis KJ in a colitis mouse model, making it suitable for publication in microbiology research.

Major Comment: The manuscript requires concise writing and contains numerous scientific and grammatical errors. Minor Comments: 1. Abbreviate LPS in line 33. 2. Relocate Table 1 to its corresponding text location. 3. Omit the sentence on line 373 or rephrase it as a suggestion for future research.

Extensive English language editing is required.

Author Response

Dear Reviewer:

First of all, we greatly appreciate your excellent suggestions. We revised our manuscript (microbiolres-2963047) according to the suggestions, and the revised sections in the manuscript are highlighted in red. I hope you will consider this paper as suitable for publication in your journal.

Again, thank you for your wonderful help.

Sincerely Yours,

authors

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Comments 1: The manuscript requires concise writing and contains numerous scientific and grammatical errors.

Response 1: Thanks for pointing this out. We agree with this opinion. Therefore, the manuscript was edited according to the editing system recommended by MDPI. I attach the relevant certificate.

Comments 2: Abbreviate LPS in line 33.

Response 2: Thank you for your comment. We revised it according to your comment (line 49).

Comments 3:  Relocate Table 1 to its corresponding text location.

Response 3: Thank you for your comment. We revised it according to your comment (line 285).

Comments 4: Omit the sentence on line 373 or rephrase it as a suggestion for future research.

Response 4: Thank you for your comment. We revised it according to your comment (line 451-453).

Author Response File: Author Response.pdf

Reviewer 2 Report

Reviewer Comments

Inflammatory bowel disease (IBD) is a multifactorial disease with an ill-defined etiology. It includes Cron’s disease and ulcerative colitis. it is characterized by impairment of the gut/liver, gut/brain, and gut/renal axes.

The article by Kim et al., entitled “Weissella koreensis KJ, which increases gut-tight junction protein expression, alleviates TNBS-induced colitis by suppressing inflammatory cytokines” represents good work on the beneficial effects of lactic acid bacteria and its anti-inflammatory mechanism of action.

Lactic acid bacteria have been found to possess anti-inflammatory effects, particularly in the management of ulcerative colitis. By modulating the immune response and reducing inflammatory cytokines, lactic acid bacteria contribute to the amelioration of colitis symptoms. This highlights their potential therapeutic role in managing IBD.   

Overall, this is an interesting work, and here are some of my comments: -

Title:

Consider rephrasing for clarity.

Abstract: The abstract lacks sufficient detail and should better summarize the study.

Include a brief introduction to IBD and its prevalence.

Expand the methods section in the abstract.

The study is therapeutic so remove the prophylaxis from the conclusion.

Introduction

The introduction is very short and insufficient, the authors should address the significance of IBD as a major health problem, its pathophysiology, names, and roles of tight junction proteins in leaky gut and impaired gut to (liver-brain- Kidney) axes.

Material and Methods

All antibody numbers should be added.

How the LAB strains were isolated from Kimchi should be mentioned.

Housing and cages of the animals should be mentioned

All abbreviations must be preceded by the full name Like ICR mice, MRS broth...etc. Please do the same for the whole manuscript.

The dose of LPS treatment is to be mentioned.

Induction of colitis is to be mentioned in detail, dose, time course, …etc.

P167: 2.9 Immunoblot Analysis and ELISA

This part is very short and must be rewritten in detail, including sample preparation, protein assay, and how much proteins (mg) are used?

For western blotting, gel %, membrane type, antibodies dilution, chemiluminescence, and detection instrument should be specified.

For Elisa mention the wavelength and the maker of the plate reader.

2.10 H&E Staining of Colon Tissue

Move the name and maker of the microscope used in the acquisition of the pictures to this section first.

For immunohistochemistry, mention the dilution of the antibodies.

2.12 Statistical Analysis

Mention the program used to statistically analyze the results with details of the maker.

Results

Figure 1 and Supplementary Figure 1

-Revise English language, grammar, and punctuation. Please do it for the whole manuscript.

Line 232- 234, rephrase and make the text color unified.

Figure 2: more histopathological morphometric changes and microscopic scores would better be included.

The western blotting analysis of B-actin is not clear

Discussion

Please write the full name of abbreviations before they appear in the text, and make it all over the manuscript.

The discussion is brief and the authors should discuss the role of leaking gut through defective Gut/(liver-brain-renal) axis in the development of IBD systemic complications

The authors would better discuss their results relative to the immune response role in the development of IBD, particularly regulatory T cells (Tregs) and T helper 17 cells (Th17).

The authors should argue for prior results that revealed increased inflammatory cytokines expression in response to LAB.

A clear conclusion should be addressed.

References

The authors should review more literature, particularly in the introduction and the discussion sections. Some references from 2000- 2008 are still there, and if possible, update with more recent relevant literature.

Moderate English Editing is required

Author Response

Dear Reviewer:

First of all, we greatly appreciate your excellent suggestions. We revised our manuscript (microbiolres-2963047) according to the suggestions, and the revised sections in the manuscript are highlighted in red. I hope you will consider this paper as suitable for publication in your journal.

Again, thank you for your wonderful help.

Sincerely Yours,

authors

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Comments 1: Title: Consider rephrasing for clarity.

Response 1: Thanks for pointing this out. We agree with this opinion. Therefore, the manuscript was edited according to the editing system recommended by MDPI. I attach the relevant certificate.

Comments 2: Abstract: The abstract lacks sufficient detail and should better summarize the study. Include a brief introduction to IBD and its prevalence. Expand the methods section in the abstract. The study is therapeutic so remove the prophylaxis from the conclusion.

Response 2: We revised it according to your comment. We added the definition and prevalence of IBD to the abstract and removed information about prevention.

Comments 3:  Introduction; The introduction is very short and insufficient, the authors should address the significance of IBD as a major health problem, its pathophysiology, names, and roles of tight junction proteins in leaky gut and impaired gut to (liver-brain- Kidney) axes.

Response 3: We revised it according to your comment (line 28-37, line 41-44), and the revised sections in the manuscript are highlighted in red.

Comments 4: Material and Methods; All antibody numbers should be added.

Response 4: We revised it according to your comment (line 83-86), and the revised sections in the manuscript are highlighted in red.

Comments 5: How the LAB strains were isolated from Kimchi should be mentioned.

Response 5: Thank you for your comment. We revised it according to your comment (line 96-99). The revised sections in the manuscript are highlighted in red.

Comments 6: Housing and cages of the animals should be mentioned.

Response 6: Thank you for your comment. We revised it according to your comment (line 164). The revised sections in the manuscript are highlighted in red.

Comments 7: All abbreviations must be preceded by the full name Like ICR mice, MRS broth...etc. Please do the same for the whole manuscript.

Response 7: Thank you for your comment. We revised it according to your comment (line 96, 144). The revised sections in the manuscript are highlighted in red.

Comments 8: The dose of LPS treatment is to be mentioned.

Response 8: Thank you for your comment. We revised it according to your comment (line 151). The revised sections in the manuscript are highlighted in red.

Comments 9: Induction of colitis is to be mentioned in detail, dose, time course, …etc.

Response 9: Thank you for your comment. We revised it according to your comment (line 175-179). The revised sections in the manuscript are highlighted in red.

Comments 10: P167: 2.9 Immunoblot Analysis and ELISA. This part is very short and must be rewritten in detail, including sample preparation, protein assay, and how much proteins (mg) are used?

Response 10: Thank you for your comment. We revised it according to your comment (line 199-211, 215-223). The revised sections in the manuscript are highlighted in red.

Comments 11: For western blotting, gel %, membrane type, antibodies dilution, chemiluminescence, and detection instrument should be specified.

Response 11: Thank you for your comment. We revised it according to your comment (line 199-211). The revised sections in the manuscript are highlighted in red.

Comments 12: For Elisa mention the wavelength and the maker of the plate reader.

Response 12: Thank you for your comment. We revised it according to your comment (line 221-223). The revised sections in the manuscript are highlighted in red.

Comments 13: 2.10 H&E Staining of Colon Tissue. Move the name and maker of the microscope used in the acquisition of the pictures to this section first.

Response 13: Thank you for your comment. We revised it according to your comment (line 229). The revised sections in the manuscript are highlighted in red.

Comments 14: For immunohistochemistry, mention the dilution of the antibodies.

Response 14: Thank you for your comment. We revised it according to your comment (line 238). The revised sections in the manuscript are highlighted in red.

Comments 15: 2.12 Statistical Analysis. Mention the program used to statistically analyze the results with details of the maker.

Response 15: We revised it according to your comment (line 248-249).

Comments 16: Results. Figure 1 and Supplementary Figure 1. Revise English language, grammar, and punctuation. Please do it for the whole manuscript.

Response 16: Thank you for pointing this out. We agree with this comment. Therefore, the paper has been edited.

Comments 17: Line 232- 234, rephrase and make the text color unified.

Response 17: We revised it according to your comment (line 280-283).

Comments 18: Figure 2: more histopathological morphometric changes and microscopic scores would better be included.

Response 18: Unfortunately, the H&E-stained images were not interpreted by a pathologist. However, it is possible to confirm the degree of edema and the degree of immune cell infiltration through the image. Symptoms of colitis shown in H&E-stained images are consistent with the macroscopic score. This information is described in lines 299-301.

Comments 19: The western blotting analysis of B-actin is not clear.

Response 19: Unfortunately, there is not enough tissue to perform blotting again using mouse colon tissue. At the bottom of Figure 4B, there is a result of analyzing the intensity of tight junction protein based on β-actin intensity. Based on these results, it seems appropriate to compare the expression of tight junction proteins.

Comments 20: Discussion. Please write the full name of abbreviations before they appear in the text, and make it all over the manuscript.

Response 20: Thank you for your comment. We revised it according to your comment.

Comments 21: The discussion is brief and the authors should discuss the role of leaking gut through defective Gut/(liver-brain-renal) axis in the development of IBD systemic complications.

Response 21: Thank you for your comment. We revised it according to your comment (line 373-381). The revised sections in the manuscript are highlighted in red. We have added discussion regarding intestinal permeability as it relates to the gut-brain and gut-liver axis and its role in influencing the pathogenesis and complications of colitis.

Comments 22: The authors would better discuss their results relative to the immune response role in the development of IBD, particularly regulatory T cells (Tregs) and T helper 17 cells (Th17).

Response 22: Thank you for your comment. We revised it according to your comment (line 410-419). The revised sections in the manuscript are highlighted in red. We added discussion that Treg/Th17 balance is very important in IBD and that regulation of Treg/Th17 by IL-6 inhibition is important in IBD treatment.

Comments 23: The authors should argue for prior results that revealed increased inflammatory cytokines expression in response to LAB.

Response 23: Thank you for your comment. We revised it according to your comment (line 388-397).

Comments 24: A clear conclusion should be addressed.

Response 24: Thank you for your comment. We revised it according to your comment (line 454-462).

Comments 25: References. The authors should review more literature, particularly in the introduction and the discussion sections. Some references from 2000- 2008 are still there, and if possible, update with more recent relevant literature.

Response 25: Thank you for your comment. Based on your comments, we have added the latest cited literature. We appreciate your understanding as some of the literature is related to experimental methods or cites traditional theories.

Author Response File: Author Response.pdf

Reviewer 3 Report

See the attached review report. 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer:

First of all, we greatly appreciate your excellent suggestions. We revised our manuscript (microbiolres-2963047) according to the suggestions, and the revised sections in the manuscript are highlighted in red. I hope you will consider this paper as suitable for publication in your journal.

Again, thank you for your wonderful help.

Sincerely Yours,

authors

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[Reviewer 3]

Comments 1: The authors explored the importance of W. koreensis in increasing tight junction protein expression in the gut by suppressing inflammatory cytokines. They also conducted the antioxidant activity of the phenolic compounds. According to the, this is a main compound. This is a flavonoid compound. Why do the authors focus on phenolic compounds if this is the case?

Response 1: Thank you for your comment. As mentioned, we studied W. koreensis, which increases intestinal tight junction protein expression by suppressing inflammatory cytokines. W. koreensis is a probiotic isolated from kimchi and is not the flavonoid compound you mentioned. Additionally, our study was not a study of the antioxidant activity of phenolic compounds.

Comments 2: 1. Abstract a. The authors could shorten the background, give values, and name the abundant phenolic structure. b. Keywords – include triglycoside as a keyword.

Response 2: Thank you for your comment. But our study was not a study of the antioxidant activity of phenolic compounds.

Comments 3:  2. Introduction a. TNBS- is it a toxin? Is it given artificially?

Response 3: Picrylsulfonic acid solution is a hapten, which can bind to tissue proteins and turn into an antigen. This leads to several immunologic responses. Colitis induced by a 2,4,6-trinitrobenzene sulfonic acid (TNBS) is a type of delayed hypersensitivity reaction to haptenized proteins. Leukotriene B4 (LTB4) and the monhydroxyl fatty acids 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE are the major inflammatory mediators involved in this TNBS-induced colitis. Ethanol when used in high concentration act as a vehicle for the TNBS administration.

     Induction of colitis was done by the intrarectal injection of a 2.5% (w/v) TNBS solution (100 μl) in 50% (v/v) ethanol into the colon of the mice. For intrarectal injection of TNBS solution, the needle was introduced about 3.5–4 cm proximal to the anus area. We revised it according to your comment (line 175-179). The revised sections in the manuscript are highlighted in red.

Comments 4: b. Why did the authors select kimchi to isolate bacteria?

Response 4: Thank you for your comment. Kimchi, a traditional Korean fermented food, contains various lactic acid bacteria (LAB), including Lactobacilli, and their health benefits have been demonstrated in various studies. This information is written in the introduction section (line 63-65).

Comments 5: c. Has any previous study been conducted on this matter?

Response 5: There are many research results on the immunomodulatory effects of lactic acid bacteria, and there are also many research results on the possibility of alleviating colitis. However, many studies show results for Lactobacillus or Bifidobacterium. We present results showing that Weissella isolated from kimchi, a traditional Korean fermented food, has the potential to alleviate immune diseases and is thus novel. This is described in the discussion (lines 447-450).

Comments 6: d. The authors should highlight the research gap of the study.

Response 6: Thank you for your comment. In this paper, we present a new IBD treatment strategy through simultaneous suppression of immune response and leaky gut and report novel lactic acid bacteria that is compatible with this treatment strategy. These contents are novel and have gap from existing research results.

Comments 7: 3. Materials & Methods a. The authors have used gastric juice as well. But nothing is mentioned in the introduction.

Response 7: Thank you for your comment. Artificial gastric juice is described in Materials and Methods 2.3 (lines 116-119). Additionally, a discussion of the results is provided in the discussion (lines 438-440).

Comments 8: b. It is not clear how the authors measured the attachment of epithelial cells.

Response 8: Thank you for your comment. We revised it according to your comment (Materials and Methods 2.4).

Comments 9: c. How did the authors assess the gut leakage after treating with TNBS?

Response 9: Tight junction protein analysis through immunoblotting and IHC were evaluated. Related information is described in discussion (line 427-429).

Comments 10: d. Expand MPO when used first.

Response 10: Thank you for your comment. Abbreviate MPO in line 82.

Comments 11: 4. Results a. Were the two species grown in the same climatic zones? The authors must indicate this. b. Are all the phenolic acids like the other species? c. Tables 1-3 include the recorded activities wherever possible.

Response 11: Thank you for your comment. But our study was not a study of the antioxidant activity of phenolic compounds.

Comments 12: 5. Discussion a. What is the purpose of conducting antioxidant assays? b. It is better to conduct an in-silico analysis of the compound structure. Check “Integration of in vitro and in-silico analysis of Caulerpa racemosa against antioxidant, antidiabetic, and anticancer activities. Sci Rep 12, 20848 (2022). https://0-doi-org.brum.beds.ac.uk/10.1038/s41598-022-24021-y” and A novel in-silico benchmarked capable of complete protein analysis: A possible tool for potential drug discovery. MDPI Biology, 2021, 10, 1113. doi:10.3390/biology10111113 c. Does this study confirm the therapeutic claims? d. Inter-relate the results. e. Highlight the relationship between TFC and antioxidant results. f. Does this study agree with previously isolated compounds? g. How effective would this flower extract be in preventing any possible diseases? h. What would be the future perspective?

Response 12: Thank you for your comment. But our study was not a study of the antioxidant activity of phenolic compounds. This study focuses on the anti-inflammatory effects of new lactic acid bacteria isolated from kimchi. MPO activity was measured, but this is only part of the anti-inflammatory effect. Although the proposed assay is considered effective for drug analysis, this appears to be outside the scope of our study, so we ask for your understanding for any inapplicability.

Comments 13: 6. References a. Check reference 35.

Response 13: We revised it according to the reference form.

Reviewer 4 Report

Dear authors

I read with interest the article Weissella koreensis KJ, which increases gut-tight junction protein expression, alleviates TNBS-induced colitis by suppressing  inflammatory cytokines, that I was invited to review.

I have some suggestions:

Line 24 how came the idea of this study?

Line 61 -I suggest the introduction of a brief description of the techniques used

Line 130- please, describe in more detail the ELISA parameters used for reading

Line 141- can you increase the studied groups at 10 mice instead of 6?

Line 343-356- don,t really sustain the utility of the study, by contrary recognize its limitations.Can you, please reformulate?

Line 377- could you, please describe in more detail what is kimchi? And how is prepared?

Author Response

Dear Reviewer:

First of all, we greatly appreciate your excellent suggestions. We revised our manuscript (microbiolres-2963047) according to the suggestions, and the revised sections in the manuscript are highlighted in red. I hope you will consider this paper as suitable for publication in your journal.

Again, thank you for your wonderful help.

Sincerely Yours,

authors

-----------------------------------------------------------------------------------------

Comments 1: Line 24 how came the idea of this study?

Response 1: Leaky gut has recently been found to be linked to a variety of diseases (Reference 1), and recent research has confirmed that this leaky gut can be controlled by lactic acid bacteria, and that suppressing them has an anti-inflammatory effect (Reference 9). Based on these previous research results, a hypothesis for this study was established (lines 71-73) and an experiment was conducted.

Comments 2: Line 61 -I suggest the introduction of a brief description of the techniques used.

Response 2: I think the important part of this study is the colitis model that includes leaky gut. The implementation of this model is described in the introduction section (lines 55-62).

Comments 3: Line 130- please, describe in more detail the ELISA parameters used for reading.

Response 3: Thank you for your comment. We added it according to your comment to the ‘2.9 Immunoblot Analysis and ELISA’ section (line 215-223). The revised sections in the manuscript are highlighted in red.

Comments 4: Line 141- can you increase the studied groups at 10 mice instead of 6?

Response 4: Unfortunately, Eulji University's IACUC is active in animal reduction. Since the experiment has already been approved and conducted with 6 animals per group, it is difficult to obtain approval for the same experiment again. We will actively consider adjusting the number of animals per group when conducting additional research in the future.

Comments 5: Line 343-356- don’t really sustain the utility of the study, by contrary recognize its limitations. Can you, please reformulate?

Response 5: Thank you for your comment. We revised it according to your comment (line 425-435). The revised sections in the manuscript are highlighted in red.

Comments 6: Line 377- could you, please describe in more detail what is kimchi? And how is prepared?

Response 6: Thank you for your comment. Kimchi, a traditional Korean fermented food, contains various lactic acid bacteria (LAB), including Lactobacilli, and their health benefits have been demonstrated in various studies. This information is written in the introduction section (line 63-65).

Round 2

Reviewer 4 Report

Dear authors

I read the final version of the manuscript and I consider the suggestions that I made were accomplished.

Thanks you and good luck

Comments for author File: Comments.docx