Anti-Inflammatory Effect of Caffeine on Muscle under Lipopolysaccharide-Induced Inflammation
Round 1
Reviewer 1 Report
In the article titled: „Anti-inflammatory effect of caffeine on muscle under lipopolysaccharide-induced inflammation” Tuany Eichwald et al. examined the effects of caffeine administration on the expression of inflammatory, adenosine receptors, epigenetics, and oxidative metabolism-linked genes in the vastus lateralis muscle of mice submitted to LPS-induced inflammation. Manuscript needs improvement:
1. in the introduction there is no description of what is the content of the publication - one sentence at the end of the introduction is not informative.
2. please change the font in the figures - for example, fig 1 is illegible (IL-1, TNFalpha)
3. please complete the description of the collection of muscle tissue, the method of killing the animals and possible storage of the collected tissue.
4. Discussion should be enriched with information about the genes studied - lacking to justify the research conducted.
Author Response
RESPONSES TO THE REVIEWER #1
Comments and Suggestions for Authors
In the article titled: „Anti-inflammatory effect of caffeine on muscle under lipopolysaccharide-induced inflammation” Tuany Eichwald et al. examined the effects of caffeine administration on the expression of inflammatory, adenosine receptors, epigenetics, and oxidative metabolism-linked genes in the vastus lateralis muscle of mice submitted to LPS-induced inflammation.
Manuscript needs improvement:
- in the introduction there is no description of what is the content of the publication - one sentence at the end of the introduction is not informative.
Response:
We extensively modified the introduction section to be more informative, and to describe better our objectives and hypothesis. As a consequence, we also added key references that were missing.
- Introduction
Regular moderate intensity exercise is proven to promote an anti-inflammatory state that helps to prevent the development of chronic diseases [for a review see (Scheffer and Latini, 2020)]. Strenuous exercise can lead to increased levels of blood proinflammatory cytokines, which are linked to fatigue, and therefore, to reduced performance (Hargreaves and Spriet, 2020). This scenario has seen caffeine administration used to increase alertness (Vital-Lopez et al., 2018), to accelerate metabolism (Harpaz et al., 2017), and to delay fatigue development in aerobic and anaerobic exercises, including muscular strength (Astorino et al., 2007), running (Graham and Spriet, 1991), cycling (Spriet et al., 1992), team sports (Schneiker et al., 2006), among others.
While it is unclear what molecular mechanisms are behind caffeine consumption and its ergogenic responses, evidence is mounting that caffeine may induce anti-inflammatory effects in both humans and animals. For example, it was demonstrated that caffeine supplementation reduced proinflammatory markers in the blood of athletes (Rodas et al., 2020; Tauler et al., 2016, 2013). In the case of animal models, reduced proinflammatory and increased anti-inflammatory markers were not only seen in the blood of trained rats, but also in key tissues linked to exercise performance, like the brain, the lung, the heart and the skeletal muscle of rodents exposed to caffeine (Barcelos et al., 2014; Endesfelder et al., 2020; Farokhi-Sisakht et al., 2022; Jia et al., 2014; Yang et al., 2022). Furthermore, an elegant study involving 114 participants showed that caffeine intake is associated with lower inflammation and activation of the inflammasome, which resulted in less production of the proinflammatory cytokyne interleukin-1 beta (IL-1ß) (Furman et al., 2017).
In addition, caffeine supplementation has also been shown to cause changes in gene expression that could be linked to improved exercise performance (Egan et al., 2013; Egan and Zierath, 2013; Seaborne et al., 2018). These modifications have been related to altered epigenetics, a term conceived to describe the possible causal processes acting on genes that regulate phenotype (Waddington, 2012). Some of the reported effects of caffeine are associated with DNA methylation, a major epigenetic factor influencing gene activities (Chuang et al., 2017). Considering that epigenetics can change the activity of a DNA segment without changing the sequence, it is plausible that caffeine can modulate inflammatory processes by changing the epigenetic landscape. When DNA methylation is increased in a gene promoter, it will typically act to repress gene transcription, including the expression of inflammatory mediators. Altogether, we aimed to understand whether caffeine can modulate epigenetics to induce an anti-inflammatory scenario in the mouse skeletal muscle.
- please change the font in the figures - for example, fig 1 is illegible (IL-1, TNFalpha)
Response:
As suggested, the font of the figures was modified to make the content of the graphs clearer (all figures were included in the revised version of the manuscript and also in the last page of this file).
- please complete the description of the collection of muscle tissue, the method of killing the animals and possible storage of the collected tissue.
Response:
Following the suggestion of the reviewer, we added the following in the Material and Methods section (page= 2; line= 83).
Twenty-four h after the treatments mice were euthanized by cervical dislocation and the vastus lateralis muscle was immediately collected and processed in Trizol as previously published by our group (remor et al 2018). The dosage of LPS used was based on previously published data (de Paula Martins et al., 2018; Ghisoni et al., 2015).
- Discussion should be enriched with information about the genes studied - lacking to justify the research conducted.
Response:
To address the issue risen by the reviewer, we added the following in the Discussion section (page= 11, line= 397)
The DNA methylation machinery requires DNMT3a and DNMT3b for the de novo (Okano et al., 1998), and DNMT1 for the maintenance (Hermann et al., 2004) of DNA methylation. In general, when methylation occurs in the promoter region of a particular gene, the gene expression is expected to be repressed. DNA can also be demethylated by the action of ten-eleven translocation (TET) enzymes TET1, TET2, and TET3 (Ito et al., 2010), which may result in enhanced gene expression. Therefore, the balance of these processes may regulate the expression of different genes, including the ones involved in inflammation and adenosine signaling as shown here. Indeed, a genome-wide meta-analysis identified several genes positively associating caffeine consumption and DNA methylation (Chuang et al., 2017; Cornelis et al., 2015, 2011). While previous studies have shown that caffeine intake positively correlated with higher DNA methylation (Chuang et al., 2017), we have shown in this study that caffeine per se can be responsible for the negative modulation of the expression of inflammatory genes in animals submitted to acute inflammation.
Finally, we would like to add that the text was thoroughly revised and edited by an English native speaker.
References cites in responses to points 1 and 4
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Author Response File: Author Response.docx
Reviewer 2 Report
This manuscript describes a study showing that epigenetics is involved in the anti-inflammatory effects of caffeine on the vastus lateralis muscle of resting mice. The results show that the treatment with caffeine prevented the increase the gene expression of LPS-induced pro-inflammatory cytokines Il1beta and Il6 and promoted the upregulation of the anti-inflammatory genes Il10 and Il13 in the mouse muscle. The experiments were carried out properly and the results were presented clearly. Beneficial aspects of caffeine have been reported in many articles so far and this report has focused on ani-inflammatory characteristics of caffeine, which is interesting. The authors should pay attention to the points below.
Issues to be considered
1 Inflammatory what? (line 6)
2 Spin should read as spun (line 116)
3 The following sentence seems to make no sense to the context and should be reconsidered.
”This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.(line 277-278)
”
4 Please explain the altered expression pattern of NLRP3 compared with those of an adaptor (Asc) and an effector (Caspase-1) (Fig. 2B). As stated in the text, although, LPS treatment per se did not alter the levels of Nlrp3 expression 24 h after the administration, the combination with caffeine provoked its upregulation. Why is it?
Author Response
RESPONSES TO THE REVIEWER #2
Comments and Suggestions for Authors
This manuscript describes a study showing that epigenetics is involved in the anti-inflammatory effects of caffeine on the vastus lateralis muscle of resting mice. The results show that the treatment with caffeine prevented the increase the gene expression of LPS-induced pro-inflammatory cytokines Il1beta and Il6 and promoted the upregulation of the anti-inflammatory genes Il10 and Il13 in the mouse muscle. The experiments were carried out properly and the results were presented clearly. Beneficial aspects of caffeine have been reported in many articles so far and this report has focused on ani-inflammatory characteristics of caffeine, which is interesting. The authors should pay attention to the points below.
Issues to be considered
1 Inflammatory what? (line 6)
Response:
We apologize to the reviewer that we could not find the issue related to inflammation in line 6. However, we noticed that the word inflammatory should have been preceded by the prefix “pro” in page=4, line= 164.
The sentence “Figure 1 shows the effect of caffeine and/or LPS administration (i.p.) after twenty-four h on inflammatory cytokines gene expression in the mouse vastus lateralis muscle (Fig. 1A).” now reads “Figure 1 shows the effect of caffeine and/or LPS administration (i.p.) after twenty-four h on pro-inflammatory cytokines gene expression in the mouse vastus lateralis muscle (Fig. 1A).”
2 Spin should read as spun (line 116)
Response:
The sentence was edited as indicated.
3 The following sentence seems to make no sense to the context and should be reconsidered.
”This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.(line 277-278)
Response:
We deleted the sentence mentioned by the reviewer (lines= 277-278). The sentence was included in the template of the journal, so it was added by mistake.
4 Please explain the altered expression pattern of NLRP3 compared with those of an adaptor (Asc) and an effector (Caspase-1) (Fig. 2B). As stated in the text, although, LPS treatment per se did not alter the levels of Nlrp3 expression 24 h after the administration, the combination with caffeine provoked its upregulation. Why is it?
Response:
The coadministration of caffeine and LPS provoked a marked reduction of Asc and Caspase-1 gene expression in the mouse muscle, pointing to an anti-inflammatory effect of caffeine. This down-regulation might have been compensated by increasing the expression of the protein that works as a receptor of the inflammasome complex. Since this is the first time the effect of caffeine under LPS treatment has been reported in the mouse muscle, we are aware that more studies are needed to better understand how caffeine induces anti-inflammatory responses.
Finally, we would like to add that the text was thoroughly revised and edited by an English native speaker.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The manuscript has been significantly revised, and is suitable for publication in its present form.