Kynurenine Is the Main Metabolite of Tryptophan Degradation by Tryptophan 2,3-Dioxygenase in HepaRG Tumor Cells

Round 1

Reviewer 1 Report

The authors have studied Trp degradation to Kyn by two key enzymes, IDO and TDO, in primary human hepatocytes (PHH) and tumoral HepG2 cells. In addition, they also investigated the relationship between Kyn accumulation and immunosuppression. However, the data can not be the direct evidence or support the conclusion. Lots of data are weak or over-presented. Therefore, I suggest the authors should have major revisions in this manuscript.

Major revision:

1.     In the introduction, the authors mentioned lots of limitations and unmet need of the therapeutic mechanism for HCC immunotherapy. However, the authors do not provide powerful and direct evidence for these questions. Suggest to revise the description in introduction and objectives or input more confident result to support the questions.

2.     In method part, the quantitation of L- and D-Trp is missing. How to detect these two isoforms? How to quantify them? How to clarify the isoform you detect or identify? Please add this information and provide the HPLC data and MS data to confirm the specific structure. These data can support Fig.7.

3.     The scale of Fig.2a is lower 10-fold than Fig.1 and Fig2b. Please explain the potential experimental deviation.

4.     In Fig.2a, it is difficult to follow in main text “Trp degradation was significantly higher in PHH cells than in HepG2 cells at 48 h and 72 h (p<0.05) (Fig. 2a)”. No information is about time course in this figure. Please correct the figure and its caption. Moreover, please explain why you can get the conclusion “This indicates that the Kyn pathway was terminated once Kyn was formed in HepG2 cells.” from Fig.2. I think the conclusion is over-presented.

5.     For Fig.3, it is also suggested to add one figure to present the ratio of Trp degradation and Kyn generation. Fig. 3b presents that high Kyn levels in the supernatants of cultured HepG2 cells within 480 mM Trp. The authors present the “…suggested that Kyn may affect TDO activity in 132 these cells.” in page 5, line 132. No result or evidence shows TDO activity in this section. Please add the reference or provide data to support this description.

6.     The name of enzyme “TMO” is misspelling. Please revise.

7.     Please check all of the spelling and spacing in the main text and revise the figure legends correctly (such as Fig. 1, Fig. 2, Fig.7). Please describe the interpretation of figure but not write the result in figure caption.

Author Response

                                                                                                                      10.08.2022

Dear Reviewers,

Thank you for your interest in our manuscript entitled “Kynurenine is the main metabolite of tryptophan degradation by tryptophan 2,3-dioxygenase in HepG2 tumor cells”. Please find below the provided responses to the Reviewer's comments. We are grateful for fulfilling all your demands and expectations. The corrections and changes have been marked up using track change in the revised manuscript. We hope that you will now find our manuscript suitable to proceed for publication in “Journal of Clinical Medicine”.

Sincerely,

Dr. Ali Ramouz

Department of General, Visceral and Transplant Surgery

Ruprecht-Karls University

Im Neuenheimer Feld 420

69120 Heidelberg, Germany

Tel: 0049-6221-5636223

Fax: 0049-6221-565781

Email: ali.ramouz@med.uni-heidelberg.de

Reviewer #1 comments  

#1: In the introduction, the authors mentioned lots of limitations and unmet need of the therapeutic mechanism for HCC immunotherapy. However, the authors do not provide powerful and direct evidence for these questions. Suggest to revise the description in introduction and objectives or input more confident result to support the questions.

Response:

Thank you for your nice comment. We updated the text to emphasize the mechanism of the tryptophan-kynurenine pathway in normal and tumor cells, as advised by the reviewer as follows (page 2; paragraph 1):

“One of the key pathways implicated in chronic liver diseases and liver malignancies is tryptophan (Trp) degradation, which leads to kynurenine (Kyn) accumulation [1-3].”

(page2; paragraph 1):

“Degradation of Trp into Kyn has the potential to stimulate tumor growth as well as influence the immune system by limiting T lymphocyte proliferation and promoting T-cell differentiation [5-7].”

(page2; paragraph 2):

“To compare the Trp-Kyn pathway in normal liver cells and tumor cells, we studied the involvement of TDO and IDO in Trp degradation and Kyn accumulation in primary human hepatocytes (PHH) and the tumoral cell line HepG2.”

#2: In method part, the quantitation of L- and D-Trp is missing. How to detect these two isoforms? How to quantify them? How to clarify the isoform you detect or identify? Please add this information and provide the HPLC data and MS data to confirm the specific structure. These data can support Fig.7.

Response:

We appreciate your suggestion. According to your comment, we have provided data on the retention time and differentiation of two distinct isoforms of Trp. All new information was included in the Method section of the paper as follows (Page 3; Paragraph 2):

„ To differentiate between two Trp isoforms, L-Trp (L-Trp) and D-Trp (D-Trp), the peaks of L-Trp and D-Trp were identified by comparison with the retention time of previously determined standard compounds, and quantification was based on the ratios of the compound's peak areas to the internal standard, with a L-Trp retention time equal to 1.197 min and a D-Trp retention time equal to 1.564 min.”  

#3: The scale of Fig.2a is lower 10-fold than Fig.1 and Fig2b. Please explain the potential experimental deviatin.

Response:

Thanks for your precious comment. As you mentioned in your comment, the scale is lower in “Fig2.a” and the reason is that in this figure, Trp degradation has been measured at 72h which is at maximum level (Minimum levels of Trp according to Figure.1). Therefore, the scale is lower 10-fold compared to other mentioned figures (Fig.1 and Fig.2b). The information about the time of measurement has been added to the caption of the “fig2.a” and the text as well.

#4: In Fig.2a, it is difficult to follow in main text “Trp degradation was significantly higher in PHH cells than in HepG2 cells at 48 h and 72 h (p<0.05) (Fig. 2a)”. No information is about time course in this figure. Please correct the figure and its caption. Moreover, please explain why you can get the conclusion “This indicates that the Kyn pathway was terminated once Kyn was formed in HepG2 cells.” from Fig.2. I think the conclusion is over-presented.

Response:

Thank you for your kind remark. As explained in response to previous comment, the measurement of Trp degradation and Kyn accumulation has been conducted at its maximum level which was at 72h. The information about the time has been corrected in the text and added to caption of “Fig.2” and the mentioned phrase “This indicates that the Kyn pathway was terminated once Kyn was formed in HepG2 cells.” was deleted from the text. as follows (Page 5; Paragraph 1):” Kyn levels were substantially greater in HepG2 cell supernatants than in PHH cell supernatants at 72 h”. 

#5:   For Fig.3, it is also suggested to add one figure to present the ratio of Trp degradation and Kyn generation. Fig. 3b presents that high Kyn levels in the supernatants of cultured HepG2 cells within 480 mM Trp. The authors present the “…suggested that Kyn may affect TDO activity in 132 these cells.” in page 5, line 132. No result or evidence shows TDO activity in this section. Please add the reference or provide data to support this description.

Response:

We appreciate your comment. As required by the reviewer, we added Trp to Kyn  ratio in different concentrations of Trp to the text and Fig.3. In addition, the mentioned phrase was deleted from the text.

#6:   The name of enzyme “TMO” is misspelling. Please revise.

Response:

According to your comment, the authors have corrected the spelling mistake.

#7:   Please check all of the spelling and spacing in the main text and revise the figure legends correctly (such as Fig. 1, Fig. 2, Fig.7). Please describe the interpretation of figure but not write the result in figure caption.

Response:

Regarding the comment, we revised all of spelling and typos errors in the main text as well as figure captions in the section of figures

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors presented the well-known immune checkpoint enzyme TDO expressed in various human cancers associated with immune evasion and tumour tolerance. Because there are few studies of TDO in hepatocellular carcinoma, the authors' experiments with PHHs and HepG2 cells seem to help advance the role of TDO as a therapeutic target in HCC.  

I have some points that I wish to be addressed. 

In Figure 3A legend, "Kyn concentrations >7 mmol/l suppress Trp degradation in HepG2 cells." -> where is it come from? 

In Figure 3B, please address an additional figure presenting the absolute values of Trp degradation differences. Also, the number in Kyn concentration on the horizontal axis seems to be rectified, according to the manuscript and the legend. 

Please correct a typo in line 145 (TMO should be rectified to TDO) and 150. 

In line 146, 680C91 concentration seems to be fixed as 40 umol/L (according to Figure 5A)

Author Response

                                                                                                                      10.08.2022

Dear Reviewers,

Thank you for your interest in our manuscript entitled “Kynurenine is the main metabolite of tryptophan degradation by tryptophan 2,3-dioxygenase in HepG2 tumor cells”. Please find below the provided responses to the Reviewer's comments. We are grateful for fulfilling all your demands and expectations. The corrections and changes have been  marked up using track change in the revised manuscript. We hope that you will now find our manuscript suitable to proceed for publication in “Journal of Clinical Medicine”.

Sincerely,

Dr. Ali Ramouz

Department of General, Visceral and Transplant Surgery

Ruprecht-Karls University

Im Neuenheimer Feld 420

69120 Heidelberg, Germany

Tel: 0049-6221-5636223

Fax: 0049-6221-565781

Email: ali.ramouz@med.uni-heidelberg.de 

Reviewer #2 comments  

#1: In Figure 3A legend, "Kyn concentrations >7 mmol/l suppress Trp degradation in HepG2 cells." -> where is it come from?

Response:

Thank you for your nice comment. As you requested, the legend figure of Fig.3a was updated as follows (Caption of Fig.3a):

“Kyn accumulation and Trp degradation for different concentration levels of Trp in HepG2 cells.”

#2: In Figure 3B, please address an additional figure presenting the absolute values of Trp degradation differences. Also, the number in Kyn concentration on the horizontal axis seems to be rectified, according to the manuscript and the legend.

Response:

We appreciate your suggestion. According to your comment, we have provided another figure which was added to Fig.3 presenting the Trp to Kyn ratio for different concentration levels of Trp. On the other hand, we changed the horizontal axis of Fig.3c to show the concentration levels of Trp and Kyn on horizontal axis of the figure in a better way.  

#3: Please correct a typo in line 145 (TMO should be rectified to TDO) and 150.

Response:

According to your comment, the authors have corrected both of spelling mistakes.

#4: In line 146, 680C91 concentration seems to be fixed as 40 umol/L (according to Figure 5A).

Response:

Thank you for your remark. As mentioned by you, the typo was corrected in the main text of the manuscript 

Author Response File: Author Response.pdf