PONE-D-21-05608

Impact of thermogenesis induced by chronic β3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteraemia in mice

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: General information:

The article is well written, the authors analyze the impact of thermogenesis induced by chronic β3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteremia in mice. In particular, there is a strong focus on the role of pro and anti-inflammatory cytokines. The authors communicate that brown and beige adipocytes activation/recruitment have a very limited impact on the host response to infection.

Strengths:

The whole article is easy to read, and the figures are clear. In addition, the objective of the study as well as the limitations of current knowledge are well highlighted. Finally results and discussion are well organized and present the main finding of the study.

Limitations:

The models used are unfortunately limited, activation of brown adipocytes and recruitment of beige adipocytes is limited to utilization of a single adrenergic agonist and the infection is limited to the utilization of a single dose of a single bacterial strain. In addition to CL316.243 utilization, authors could expose mice to cold which is the main brown/beige fat activator. In addition to this gain of function, a loss of function approach based on utilization of knock out mice (UCP1-KO for example) mice could have been considered.

Minor comments:

• In the second paragraph of the introduction the authors discuss a different response of beige and brown adipocytes after in vitro stimulation with LPS: «Interestingly, it has been shown that brown and white adipocytes respond differently to in vitro LPS stimulation and mediate different inflammatory responses (10)». A sentence explaining what the differences are would be appreciated.

• In the second paragraph of the introduction, it is mentioned that after activation of brown and recruitment of beige adipocytes, mice infected with LPS exhibit a higher pro-inflammatory response characterized by increased secretion of IL-1RA «We recently demonstrated that after recruitment and activation of brown and brite adipocytes, mice displayed a higher pro-inflammatory response when they are exposed to lipopolysaccharides derived from E. coli bacteria (11). Especially, these mice secreted high level of interleukin-1 receptor antagonist (IL-1RA) known to counteract interleukin 1β (IL-1β) inflammatory action (11)». This affirmation seems to be counterintuitive with the data presented in the paper and highlighted in the summary: “our results showed that in response to bacteria, thermogenic activity promoted a discrete and local anti-inflammatory environment in white adipose tissue characterized by the increase of the IL-1RA secretion”. Clarification is required on this matter.

• In Figure 1 and Figure 2, the authors presented an adjusted rectal temperature. However, in the material and method section the rectal temperature measurement and the adjustment are not mentioned. An explanation is required on this matter.

Major comments:

• In Figure 1, effects of CL316.243 a well-known browning inducer and brown adipocyte activator are assessed by measurement of temperature, weight loss, and lipid droplets size. This characterization could be more complete. For example, utilization of qPCR targeting characteristic beige and brown adipocytes genes (in particular the uncoupling protein UCP1) could provide a quantitate evaluation of browning/brown fat activation. In addition to the qPCRs, western blot or imaging (similar to those performed in Figure 5) could assess protein levels. Finally, because CL316.243 is an adrenergic agonist, it is lipolytic inducer. Then the size of lipid droplets is not the ideal parameter to evaluate the activation/recruitment of beige and brown adipocytes.

• In Figure 2B, the initial bacteremia seems to be lower (about 25%) in mice treated with CL316.243 compare to control mice although not significant due to the standard deviations. Thus, if we consider the slope of the graph (which corresponds to the bacteremia reduction rate), it is lower in the treated mice than in the untreated mice. So, should the authors reconsider the conclusion based on the slope? To avoid any doubt, it would be good to have an identical initial bacteremia, and if possible, to increase the number of measurements.

• In Figure 5: the authors use histology to characterize the infiltration of immune cells into subcutaneous adipose tissue. Although informative, these techniques do not provide a global overview of the whole tissue. I think that cytometry utilization after isolation of stromal vascular fraction could provide quantitative data about the different immune populations present in the tissue. In addition, it would be interesting to analyze this immune infiltration after infection in kinetic and in brown adipose tissue.

• Finally, several questions remain unanswered, and it would be interesting to answer or discuss them. Firstly, the role of beige and brown adipocytes has been studied here following a global infection, induced by the systemic injection of E.coli (strain UTI89, 2x107 CFU/ mouse). Do beige and brown adipocytes have an effect in response to local infection, directly in the tissue itself or in adjacent tissues? Secondly, the authors monitored mice during 48 hours after injection. This timing make sense because it is the time necessary for the organism to reduce the bacteremia to 0 (Cf figure 2B). However, this timing being pretty short, it would be interesting to determine the role of these beige and brown adipocytes following a long-term infection. Thirdly, a single dose of E.coli has been used in this publication, it could be interesting to determine if, face to a larger or even lethal infection, brown and beige adipocytes functions would not be exacerbated and thus more obvious. A survival curve in response to a lethal infection perform in CL316.243 treated or untreated mice could be an interesting addition.

Conclusion:

Based on the presented work I think that, although convincing, the data are not, at this stage, complete enough to be published in PLOS One.

Reviewer #2: In this paper, Munro et al investigate the inflammatory response of adipose tissues to bacterial infection in mice that were pre-treated with a B3-adrenergic agonist, inducing activation of brown adipose tissue (BAT) and browning of white adipose tissue (WAT). BAT activation and/or WAT browning were previously shown to prevent or alleviate metabolic disorders related to obesity such as insulin resistance and cardiovascular diseases. Interestingly, as mentioned by the authors, obesity has been associated with endotoxaemia consecutive to alterations in the intestinal barrier. Endotoxaemia results in local adipose tissue inflammation and dysfunction, thereby contributing to obesity-induced metabolic disorders. Hence, it is of importance to determine whether BAT activation and/or WAT browning may improve metabolic profile through modulation of the inflammatory response of adipose tissues.

The findings reported here show no alteration of the inflammatory response in adipose tissues of mice treated with the B3-agonist compared to vehicle, suggesting that activation of adipose thermogenesis does not play a major role in acute infection. Noteworthy, this paper excludes a role for leptin in induction of fever as leptin secretion decreases while body temperature increases in both non-treated and B3-agonist treated mice.

Although the results mostly show no differences between the experimental groups, this work is of interest as it disproves the hypothesis of a role for BAT activation/WAT browning in the response to acute systemic infection. Importantly, Munro et al measured inflammatory cytokines actually secreted by different adipose depots and not only mRNA levels (as usually done in most papers), strengthening the physiological relevance of this study.

Major concerns:

1/ The efficiency of B3-agonist treatment is only assessed by the morphological changes observed in adipose tissues (Figure 1E). As browning is not a homogeneous process within one adipose depot, the authors have to provide molecular data showing upregulation of classical browning markers and thermogenic genes in different adipose depots studied.

2/ The absence of significant differences in cytokine production by adipose tissues between NaCl- and CL-treated animals could be partly due to the heterogeneity of the values obtained within each group. In line with comment 1, correlations between molecular markers of BAT activation or WAT browning and production of cytokines by the different depots could be informative.

3/ Figure 2B: although there are no statistical differences between NaCl- and CL-treated mice, bacteremia seems to be different and/or heterogeneous, especially at the first timepoint. Such difference has to be commented, as well as its potential effects on the different inflammatory parameters measured subsequently.

4/ Adipose tissues comprise numerous cell types including adipocytes and immune cells, both of which could contribute to cytokine secretion. On one hand, cytokine production was measured from BAT and WAT samples; therefore, it cannot be excluded that the absence of differences between NaCl- and CL-treated animals observed at the tissue level actually reflects the contribution of immune cells, which could mask a differential response of the adipocytes at the cellular level. On the second hand, WAT browning has been associated with changes M1/M2 macrophages relative amount, which is expected to result in differences in cytokine production upon B3-agonist treatment. These two points should be discussed.

Minor concerns:

1/ In the materials and methods, it is not clear whether the CL treatment was stopped when bacteria were injected or whether it was prolonged for 2 more days until mice were sacrificed.

2/ Page 8, line 3: authors mention an increase in fatty acid oxidation inferred from a decrease in plasma TG. This is over-interpretation and has to be toned down.

3/ Pages 12-13: results show higher IL-1RA secretion by WAT after CL treatment, not by brite adipocytes (as WAT is not only made of adipocytes).

4/ The whole manuscript has to be re-checked for typos (CL 312,243/CL 316,243,...) and proper English grammar (shown/showed,…).

Reviewer #3: In this study, the authors evaluate the impact of thermogenesis on infectious response during bacteremia in mice. After one week of treatment with a β3-adrenergic receptor agonist (CL316,243), they induce acute bacterial infection with E. coli and they show an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes. These results are in accordance with a previous study in which the authors demonstrated an IL-1RA anti-inflammatory response to LPS after activation of brown adipose tissue. However, in the present study, the authors do not show an alteration in adipose tissue and a limited pro- and anti- inflammatory cytokines production. These discrepancies between LPS and bacteria infection could be explained by the hypothesis of “an innate immune function of the adipose tissue in chronic rather than in acute infections”. Despite of the limited impact of this work because of some negative results, the use of bacterial infection is more physiological relevant to trigger sepsis than the use of LPS and this study open the way to further works. It highlights the importance to perform a chronic rather than an acute infection to evaluate the impact of adipose tissue on bacterial infection in a physiological context.

Study design is well described and results are clearly analysed and discussed but there are minor concerns regarding some mistake and points that should be clarified:

1/ There are some mistakes in the legend of the figure, notably regarding the number of samples:

• Figure 1: n = 12 -16 or 8 (A) or 6- 8 (B-D)

• Figure 2: n = 6 (mice) or 8 -4 (scWAT explants). The authors forgot to mention n=8 (plasma)

• Figure3: There is a mistake in the legend: “IL-2 levels were assessed in the plasma (A) or in the media of iBAT (AB) and scWAT (BC)”. n = 6 -8 (plasma) or 8 - 4(explants).

• Figure 4: n = 6 (plasma) or 8 -4 (explant).

• Figure 5: The authors have to mention the number of experiments represented by these pictures.

• Figure 6: n = 6- 8 (plasma) or 8 or 4 (explant).

More generally, can the authors explain why the “n” is not the same between plasmas and explants analysis?

2/ Introduction, page 3: there is a mistake, the authors mention that “mice displayed a higher pro-inflammatory response” instead of “anti-inflammatory response”.

3/ Fig 2A: Unlike the authors say that “the fever in response to bacteria is equivalent between NaCl- and CL316,243-treated mice”, this must be confirmed. As the rectal temperature is different before infection, the authors should normalize all the values to the initial value and to comment the increase induced by the infection. It seems evident that during the first 4 hours post infection, the increase is higher in the NaCl group than in the other one.

4/ Fig 3B: The decrease on IL-6, IL-12, KC/GRO seems due to the fact that only 4 mice have been analysed. If we compare with the analyses in plasma, we can observe that for these same cytokines, we distinguish a heterogeneity between two groups of 4 mice. Therefore, if the authors have selected the 4 mice that was lower in plasma cytokines to analyse iBAT, they highlight a decrease which is not very right for all the group samples. It seems that we can observe the same profile in scWAT even if the decrease is no significant. Can the authors explain this point?

The 3.3 and 3.4 title are similar, is-it a mistake?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Saint-Laurent Celine

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Impact of thermogenesis induced by chronic β3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteremia in mice

PONE-D-21-05608R1

Dear Dr. Pisani,

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Fulvio D'Acquisto, PhD

Academic Editor

PLOS ONE

Reviewers' comments: