Autophagy Mediates Escherichia Coli-Induced Cellular Inflammatory Injury by Regulating Calcium Mobilization, Mitochondrial Dysfunction, and Endoplasmic Reticulum Stress

Round 1

Reviewer 1 Report

This is a good concise paper.

However it is only speculation, it is not valued to read.

Author Response

Please see the attachment.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The article and the topic are very interesting. The manuscript is generally well-written and well-structured. Nevertheless, the authors should improve the discussions section by comparing their results with other results reported in the literature. In this form, the authors used mostly general information in the discussions section that should be removed since they do not represent the aim of the discussions section.

Moreover, the conclusions should focus more on highlighting the clinical relevance of this study.

Author Response

Dear Reviewer:

 

Thank you very much for the helpful comments on our manuscript (ID: ijms-1971059). We have read the comments carefully and have adjusted the manuscript accordingly. Our response to your specific comments are outlined below.

 

A revised manuscript has been uploaded to the journal website. Please feel free contact us with any further questions.

 

Best Regards,

 

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Comments and Suggestions for Authors: The article and the topic are very interesting. The manuscript is generally well-written and well-structured.

 

Point 1: Nevertheless, the authors should improve the discussions section by comparing their results with other results reported in the literature. In this form, the authors used mostly general information in the discussions section that should be removed since they do not represent the aim of the discussions section.

 

Response 1: Thank you very much for your suggestion. We have improved the discussion section of the manuscript by comparing our results more fully with the reports in the literature and have removed more general information in the discussion section.

 

Point 2: The conclusions should focus more on highlighting the clinical relevance of this study.

 

Response 2: Thank you very much for your suggestion. We have rewritten the conclusions in the manuscript as follows: In this study, we demonstrated that autophagy is a key regulatory pathway in the inflammatory response caused by E. coli in primary bEECs. Restoring autophagic functions can effectively attenuate the inflammatory damage response. These observations are of particular significance for reducing the duration of bovine postpartum endometritis and for promoting rapid postpartum pregnancy.

Reviewer 3 Report

This study is being validated using cultured cells as a model system for cellular response to bacterial infection in endometritis. This reviewer thinks that this article is worth for publication after several modifications. This reviewer would like the authors to consider the following points;

 

(1)  How does the cellular response change in response to Multiples of infections (MOI)? Is a 10-fold increase in the number of E. coli relative to the number of cells (MOI 10) tested in this paper considerably more excessive conditions than is actually the case. How about the cellular response attenuated at MOI 0.1 and 1? Is there a response threshold?

 

(2)  EGTA, NAC, BAPTA-AM, and 2-APB treatment experiments should be checked for response to bEECs not infected with E. coli as control experiments.

 

(3)  Please add explanations for the abcd annotations in the graphs to the figure legends.

Author Response

Dear Reviewer:

 

Thank you very much for the helpful comments on our manuscript (ID: ijms-1971059). We have read the comments carefully and have adjusted the manuscript accordingly. Our response to your specific comments are outlined below.

 

A revised manuscript has been uploaded to the journal website. Please feel free contact us with any further questions.

 

Best Regards,

 

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Comments and Suggestions for Authors: This study is being validated using cultured cells as a model system for cellular response to bacterial infection in endometritis. This reviewer thinks that this article is worth for publication after several modifications. This reviewer would like the authors to consider the following points;

 

Point 1: How does the cellular response change in response to Multiples of infections (MOI)? Is a 10-fold increase in the number of E. coli relative to the number of cells (MOI 10) tested in this paper considerably more excessive conditions than is actually the case. How about the cellular response attenuated at MOI 0.1 and 1? Is there a response threshold?

 

Response 1: We are sorry that this problem has confused you. In fact, we screened the multiplicity of infection (MOI) of primary bovine endometrial epithelial cells (bEECs) infected with E. coli at an early stage of the study. We consulted a wide range of the literature as there are numerous differences in MOI for different cell lines, such as bone marrow-derived macrophages (BMDM) and peritoneal macrophages (MOI, 0.2, 0.5) [1], intestinal porcine epithelial cell line 1 (IPEC-1) cells (MOI, 0.1) [2], sheep endometrial epithelium cells (MOI, 1) [3], bovine mammary epithelial cells (MOI, 5) [4], and Caco-2 cells (MOI, 10) [5-7]. Some papers even report E. coli MOI of 50 or 100 for cell infection [8]. The MOI gradients we selected at the initial stage were 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1 and 100:1. However, changes in cellular morphology gradually appeared as the MOI increased, and significant changes in morphology were observed when the MOI of E. coli was 20:1, 50:1, and 100:1 at which point vacuolization, shrinkage, transparency, detachment, and even dead cells appeared. The expression of NF-κB, IL-1β, and IL-18 increased with a dose-dependent effect when MOI was below 10:1, and decreased gradually when MOI exceeded 10:1. These results indicate that the MOI of E. coli at 10:1 was appropriate to establish the inflammation model of primary bEECs. Since the cells we used were primary bEECs, which may be less prone to infection than cell lines such as RAW264.7, a higher MOI was possible. Concerning a response threshold, the primary bEECs infected with E. coli indeed have a response threshold. In our study, E. coli infection significantly activated the inflammatory response at a MOI of 1 for 3 h.

 

Point 2: EGTA, NAC, BAPTA-AM, and 2-APB treatment experiments should be checked for response to bEECs not infected with E. coli as control experiments.

 

Response 2: Thank you very much for your suggestion and we are sorry that this problem has confused you. The inhibitors we used in the study were commercially available and numerous previous studies have demonstrated their function [9-12]. As the aim of the present study was to explore whether the inhibitors exerted effects during E. coli infection, we did not include groups only treated with the inhibitors.

 

Point 3: Please add explanations for the abcd annotations in the graphs to the figure legends.

 

Response 3: Thank you very much for your suggestion. We have added explanations for the abcd annotations in the figure legends in the manuscript.

 

 

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Round 2

Reviewer 1 Report

: However it is only speculation, it is not valued to read.

Author Response

Comments: However, it is only speculation, it is not valued to read.

Point: Thank you for your comments and wish you a happy life.

Reviewer 3 Report

I have no further questions. The paper is of a sufficient level to be published and I recommend its acceptance.

Author Response

Comments and Suggestions for Authors：I have no further questions. The paper is of a sufficient level to be published and I recommend its acceptance.

 

Point: Thank you for your comments and wish you a happy life.