Better In Vitro Tools for Exploring Chlamydia trachomatis Pathogenesis

Reviewer 2’s Comments

Authors’ Answers

The article is rather short but dedicates most space to background information that appears unnecessary for the discussion of the topic at hand, while the topic itself is hardly touched upon at all. This background information, an extended summary of our current knowledge of how Chlamydia interacts with host cells, is in some parts also erroneous or outdated. It is unclear why some aspects are explained with much detail and others are omitted.

As suggested by the Reviewer, we revised and shortened the paragraph on C. trachomatis (see page 2-3, lines 59-119).

The description of available culture models remains superficial. Examples of different systems are given, but what is missing is a comprehensive description of systems available and a clear description of their characteristics, advantages, and limitations. We are clearly not limited to HeLa cells, McCoy cells, Sertoli cells, and organoid, as the article somehow would suggest. A major question that should be addressed is also in what ways our different systems can mimic the natural infection conditions and in what ways they fail to do so. Which questions can be answered in cell cultures and which questions require cultures of primary cells, polarized cells, organoids, or ex vivo fallopian tubes; and why? Which questions can to date only be addressed in cell lines? Which questions still require the use of animal models?

As suggested by the Reviewer, we revised the whole manuscript, including the description of the available systems and their characteristics, advantages and limitations as well as how the available systems can better mimic the natural infection conditions and their implications (see page 3-7, lines 120-213, and table 1).

What also appears to be missing is a perspective for the future. What new knowledge are we expected to gain through the use of better or alternative models? Are there any additional alternatives that the field has not explored yet (e.g. organ-on-a-chip technology or else)?

We have revised the discussion section addressing the Reviewer’s concerns and adding the organ-on-a-chip technology (see page 7, lines 174-213).

The figure is not helpful, as it only describes the origin of cells used in previous organoid cultures, which could instead be mentioned in the text. Instead a figure could be added that describes and compares the characteristics of the different systems.

We thank the Reviewer for the suggestion, and we have replaced the Figure 1 with a Table summarizing the main characteristics, advantages and limitations of the different systems (see table 1, page 5-6).

The authors mention that there is currently renewed interest in studying C. trachomatis. I feel unsure why this is. Was there a time where interest was lost?

We are sorry for the misunderstanding; we have rephrased the statement at page 7, lines 174-181.

Reviewer 3’s Comments

Authors’ Answers

Line 8-10: Reframe the statement mentioning study rationale in more clear and precise language and support the statement with recent epidemiological/demographical data set, if available.

We have re-written the statement as suggested by the Reviewer (see page 1, lines 8-12).

Please capitalize the first letter of the word “Several”.

We capitalized the word “Several” (see page 1, lines 13).

Line 30: Grammar correction: Use “if left Untreated/ untreated infections” whichever is appropriate.

We revised the grammar mistake as suggested by the Reviewer (see page 1, lines 33-34).

Line 44-46: Repetition: Written the same statement as inline 14-16.

We reported in the Abstract the same aim of the study than that reported in the manuscript for avoiding misunderstandings.

Please rewrite the 104-106 lines in a more meaningful sentences. Change: Recently, further…. 

We have revised the sentence as suggested by the Reviewer (see page 3, lines 100-102).

Please reframe Line 121-122: Grammar Correction-Subsequently causes inflammatory cell death.

We have shortened the paragraph concerning C. trachomatis, and, hence, the statement “subsequent inflammatory cell death” has been removed, as suggested by the Reviewer 2 (see page 3, lines 116-119).

Line 129-131 – Rewrite the sentence to define organoids and explain its advantages in the disease/infection model. Please mention the basic unit of organoid architecture is Stem cells, resulting in Mini-Organ.

We have re-organized the description of the currently available 3D cell culture models, expanding on the characteristics, advantages and limitations of the actually available systems as suggested by the Reviewer 2 (see pages 3-4, lines 120-169).

Figure 1: Check if the figure is matching with statements 134-135. First model was developed on murine endometrial primary cells whereas the arrow in figure shows human endometrium that was used for developing the second model. Please resolve this confusion using separate diagrams for each model, possibly.

We have replaced Figure 1 with a Table summarizing the characteristics, advantages and limitations of the available systems (see Table 1, pages 5-6).

Please remove doubled words, In future from line No. 213.

We have revised the statement (See page 7, lines 210-213).

General comment: Please briefly mention about currently available advanced microscopic, immunological and molecular techniques to explore C. trahomatis pathogenesis. The advantages and disadvantages of these techniques would support this article and help in making it complete.

We agree with the Reviewer and we have briefly mentioned the suggested aspects in different parts of the manuscript.