Effects of Caffeine and 5-Caffeoylquinic Acid on Blood Cell In Vitro Cytokine Production in Response to Lipopolysaccharide Stimulation

Round 1

Reviewer 1 Report

Dear Editor of Applied Science, 

The article entitled "Effects of caffeine and 5-cafeoylquinnic acid on blood cell in vitro cytokine production in response to lipopolysaccharide stimulation" is intersting. The article may be accepted for publication after improving the materials and methods, particularly in the following points:

1. The procedure to prepare the samples containing caffeine and TCA is not clear.
2. How is the participants consume the sampels? The procedure must be well-explained.  

Author Response

The article entitled "Effects of caffeine and 5-cafeoylquinnic acid on blood cell in vitro cytokine production in response to lipopolysaccharide stimulation" is intersting. The article may be accepted for publication after improving the materials and methods, particularly in the following points:

1. The procedure to prepare the samples containing caffeine and TCA is not clear.
2. How is the participants consume the sampels? The procedure must be well-explained.

We would like to thank the reviewer for the comments and suggestions. In response to both questions, it should be clarified that this is a study focused on the in vitro effects of caffeine and 5-CQA. Therefore, participants did not consume any supplementation. Instead, whole blood was collected from participants in their basal or habitual conditions and these blood samples were then used for experiments performed in the lab at an in vitro level in which the blood was incubated with solutions of caffeine 5-CQA, or LPS for example.

In this sense, caffeine and 5-CQA were added, as it is described in the manuscript (lines 116-128) to plate wells to complete the whole design of the study. Caffeine and 5-CQA were previously dissolved in culture media to reach the adequate concentrations, and then added to the wells together with blood and culture media.

Reviewer 2 Report

This paper is interesting, suggesting that caffeine and 5-cafeoylquinnic acid (5-CQA) may not affect cytokine production at physiological concentrations. However, I think you need to be more cautious in order to prove that it does not. Do you have data on IL-1β, -6, -10, -12 and TNF-α in cells not stimulated with LPS (LPS(-))? Do you also have positive control data with drugs that inhibit interleukin production, e.g. dexamethasone, and antioxidants that affect MDA production? Please add these data to clearly indicate the validity of your experimental procedure.

P6, Line 245-249: I do not understand what you say. Do you want to compare the caffein concentration showed non-significant downward trend?

P7, Line 292-293: Please confirm. Because 5-CQA at 2 microM suppressed IL-6 production significantly.

Author Response

This paper is interesting, suggesting that caffeine and 5-cafeoylquinnic acid (5-CQA) may not affect cytokine production at physiological concentrations. However, I think you need to be more cautious in order to prove that it does not. Do you have data on IL-1β, -6, -10, -12 and TNF-α in cells not stimulated with LPS (LPS(-))? Do you also have positive control data with drugs that inhibit interleukin production, e.g. dexamethasone, and antioxidants that affect MDA production? Please add these data to clearly indicate the validity of your experimental procedure.

We would like to thank the reviewer for the comments and suggestions. Unfortunately, we do not have positive control data with drugs that inhibit interleukin production and antioxidants that affect MDA production.

As it is indicated in the manuscript (lines 122-125 in the revised version), 50 microL of LPS solution or the same volume of culture media (spontaneous production) were added to cells. Following the format of previous similar studies and to include only the most relevant information, in the original version of the manuscript, only the difference between cytokine concentration in stimulated and unstimulated cultures was reported in tables 2 and 3. In the revised version of the manuscript, cytokine concentrations in cells not stimulated with LPS and in the presence of caffeine or 5-CQA are reported in supplementary tables 1 and 2. This has been indicated in lines 173 and 202, where “results not shown” was deleted and the proper table has been referenced. To allow a proper comparison, the values per cell number were maintained. Concentrations observed basically correspond to the common ones in plasma.

P6, Line 245-249: I do not understand what you say. Do you want to compare the caffein concentration showed non-significant downward trend?

In the study by Horrigan et al. a significant effect of caffeine decreasing TNF-alpha production was reported when the 100 mM caffeine concentration was considered. However, a non-significant decrease was observed for the 10 mM caffeine concentration. Therefore, our comment is in this line. In both, the present and the one from Horrigan, lower caffeine concentration tended to decrease TNF-alpha production, but not up to a significant extend.

P7, Line 292-293: Please confirm. Because 5-CQA at 2 microM suppressed IL-6 production significantly.

We would like to thank the reviewer for pointing out this mistake. However, we feel that the sense of the discussion does not change as effects are only observed for micromolar 5-CQA concentrations, values much higher than the physiological ones.

Reviewer 3 Report

Please check spelling (e.g. caffeoylquinic instead of cafeoylquinic).

In general, I do not like the term 'decrease in production'. Maybe the authors can use an alternative term such as e.g. 'the procution of xx was less pronounced'.

There is a strange symbol (@M) instead of µM (lines 58, table 3 ff) Please modify.

Sometimes I would suggest to change the wording: 'decreased stimulated production of xxx'. I suggest to write: 'reduced production' instead.

Line 41: less insted of lower

line 111: all participants' blood

Table 1: change 'stature' into 'hight'

Lines 263-277: https://doi.org/10.1017/S0007114514002232

Line 312: Please check.

Reference 4 is rather old. Please cite Phenol-explorer (Neveu et al. 2010).

Please check your references. Some of them are old and new findings were reported in the last years.

Author Response

We would like to thank this reviewer for the comments and suggestions.

Please check spelling (e.g. caffeoylquinic instead of cafeoylquinic).

We would like to thank the reviewer for highlighting this mistake. It has been corrected throughout the manuscript.

In general, I do not like the term 'decrease in production'. Maybe the authors can use an alternative term such as e.g. 'the procution of xx was less pronounced'.

We have taken on board this perspective and have amended the wording of such descriptions accordingly.

There is a strange symbol (@M) instead of µM (lines 58, table 3 ff) Please modify.

This has been corrected.

Sometimes I would suggest to change the wording: 'decreased stimulated production of xxx'. I suggest to write: 'reduced production' instead.

As above this has been done through the manuscript.

Line 41: less insted of lower

This has been amended.

line 111: all participants' blood

Amended.

Table 1: change 'stature' into 'hight'

We believe the term stature is the accurate term to use in this instance, as this term defines the measured natural height of an individual when standing upright, whereas height refers to the distance from the base of something to the top i.e. relative to the viewer.

Lines 263-277: https://doi.org/10.1017/S0007114514002232

We would like to thank the reviewer for suggesting this reference. The main conclusion of this study was introduced in the manuscript (lines 281-285).

Line 312: Please check.

We would like to thank the reviewer for highlighting this typo. It has been corrected.

Reference 4 is rather old. Please cite Phenol-explorer (Neveu et al. 2010).

This reference has been change following the suggestion of the reviewer.

Please check your references. Some of them are old and new findings were reported in the last years.

Please note that some of the oldest references are the ones reporting similar experiences to the one of the present study and are seminal works for the topic. With the inclusion of the references previously indicated by this reviewer, more recent important information has been included in the manuscript. Furthermore, we have revised and updated some of the older references where appropriate.

Round 2

Reviewer 3 Report

Thanks for taking up my suggestions.