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  1. Author Response:

    Reviewer #1:

    Bandyopadhaya et al have sought out to elucidate the immunometabolic mechanisms of monocyte tolerance induced by 2-AA, a quorum-sensing signal that is produced by Pseudomonas aeruginosa. An interesting topic, since elucidating how p. aeruginosa escapes the immune system could be very relevant from a clinical perspective.

    In previous publications, they showed that 2-AA can induce immune tolerance, leading to decreased cytokine production and epigenetic changes mediated via increased HDAC activity. In this follow-up paper, they tried to elucidate what immunometabolic changes are observed in 2-AA tolerized cells (both mouse and human cell lines) and how this can explain the improved intracellular survival of P. aeruginosa.

    The authors must be praised for the effort they put in to proof their point. They have undertaken a tremendous amount of experiments and measurements with so many different cell lines, stimuli, inhibitors and readouts. Unfortunately, the amount of figures and data also makes it very confusing and hard to read and in my opinion, they draw the wrong conclusions from the results of the experiments. Therefore, I cannot agree with some of the important statements, for example that 2-AA induces a Warburg effect. In addition, the methods are written in such a limited way, that it is hard to conclude if their conclusions are correct or to repeat these experiments.

    We thank the reviewer for their constructing comments and for appreciating the complexity of the study. We apologize for the brevity of material and methods. We hope that in addition to the data already presented, our revised manuscript will thoroughly address this reviewer’s concern on whether the Pseudomonas aeruginosa MvfR-regulated small molecule, 2-AA, indeed promotes a “Warburg-like” metabolic reprogramming in macrophages. The additional ongoing experiments, including seahorse studies and more detailed information in the materials and methods section of our manuscript, should ease this reviewer’s concerns.

    Reviewer #2:

    In the manuscript "Immunometabolic hijacking of immune cells by a Pseudomonas aeruginosa quorum-sensing signal" the authors studied the mechanism by which the quorum sensing signal 2-aminoacetophenone (2-AA), produced by the pathogen Pseudomonas aeruginosa, enables persistence of this pathogen in host tissues.

    Lactate, the fermentative product of glycolysis, reflects glycolytic fluxes and represses immune signaling activation decreasing inflammation in macrophages. Therefore, lactate levels reflect the metabolic status of the cells and has consequences for the inflammatory levels of the cells.

    In this study the authors show that 2-AA can affect the metabolic state of macrophages by increasing the glycolytic flux with the consequent increase in lactate levels and decrease in TCA flux. They also show that lactate decreases inflammation by suppressing 2-AA activation of NF-kBeta signaling and proinflammatory cytokine production.

    Using a murine model they show that addition of that 2-AA in mice infected with Pseudomonas aeruginosa results in an increase production of lactate and decrease of ATP in mice tissue, thus providing for 2-AA-mediated metabolic changes in vivo.

    The study described here is well written and the conclusions are generally well supported by the data. While they tested the direct effect of the 2-AA signal in macrophages, this was not tested in vivo in the absence of infection, and I think it is important to address the direct impact of the signal on the host.

    The study reported here proposes that a quorum sensing signal has an impact in pathogen persistence through immunometabolic reprograming properties, and provides evidence for a novel mechanism by which bacteria use quorum sensing signals to persist in the host.

    We thank the reviewer for appreciating our work, the experimental strategy, and the conclusions. We agree that the proposed additional 2-AA in vivo experiments in the absence of infection will further strengthen the in vitro studies. Additionally, they will corroborate our previously published in vivo studies on the immune responses triggered by 2-AA in absence of infection (Bandyopadhaya et al., PLoS Pathogens 2012 & Bandyopadhaya et al., Nat Microbiology, 2016).

    Reviewer #3:

    Tolerance in macrophages involves a global transcriptional shift from a pro-inflammatory response toward one characterized by the expression of anti-inflammatory and pro-resolution factors. In the case of TLR-mediated tolerance, pro-inflammatory cytokines are not universally suppressed in all tolerant cells, but distinct patterns of cytokine expression distinguished TLR-specific tolerance. (10.3389/fimmu.2018.00933, 10.1615/critrevimmunol.2015015495). However, the authors only show differences in TFNa. Thus, I strongly suggest the authors to determine anti-inflammatory cytokines, such as IL-10.

    We appreciate the reviewer’s comment and thank the reviewer for the suggestion to determine the levels of the anti-inflammatory cytokine IL-10. Indeed, we could not detect IL-10 in 2-AA tolerized cells; we will refer to this in the revised manuscript. Most likely because, as we previously demonstrated, 2-AA-mediated tolerance is markedly different from LPS mediated tolerance (Bandyopadhaya et al., PLoS Pathogens 2012 & Bandyopadhaya et al., Nat Microbiology, 2016) and TLR-regulated tolerance is primarily LPS mediated. In the previous publication (Bandyopadhaya et al. PLoS Pathogens 2012), we have also reported the IFN and anti-inflammatory TGF levels in 2-AA tolerized mouse macrophages, but we could not detect IL-10.

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  2. Evaluation Summary:

    In a previous study the authors showed that the quorum sensing signal molecule 2-aminoacetophenone (2-AA) produced by Pseudomonas aeruginosa enables persistence in host tissue of this pathogen. They propose that this effect depends on a Warburg-like metabolic reprogramming effect in macrophages.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Bandyopadhaya et al have sought out to elucidate the immunometabolic mechanisms of monocyte tolerance induced by 2-AA, a quorum-sensing signal that is produced by Pseudomonas aeruginosa. An interesting topic, since elucidating how p. aeruginosa escapes the immune system could be very relevant from a clinical perspective.

    In previous publications, they showed that 2-AA can induce immune tolerance, leading to decreased cytokine production and epigenetic changes mediated via increased HDAC activity. In this follow-up paper, they tried to elucidate what immunometabolic changes are observed in 2-AA tolerized cells (both mouse and human cell lines) and how this can explain the improved intracellular survival of P. aeruginosa.

    The authors must be praised for the effort they put in to proof their point. They have undertaken a tremendous amount of experiments and measurements with so many different cell lines, stimuli, inhibitors and readouts. Unfortunately, the amount of figures and data also makes it very confusing and hard to read and in my opinion, they draw the wrong conclusions from the results of the experiments. Therefore, I cannot agree with some of the important statements, for example that 2-AA induces a Warburg effect. In addition, the methods are written in such a limited way, that it is hard to conclude if their conclusions are correct or to repeat these experiments.

    Read the original source
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  4. Reviewer #2 (Public Review):

    In the manuscript "Immunometabolic hijacking of immune cells by a Pseudomonas aeruginosa quorum-sensing signal" the authors studied the mechanism by which the quorum sensing signal 2-aminoacetophenone (2-AA), produced by the pathogen Pseudomonas aeruginosa, enables persistence of this pathogen in host tissues.

    Lactate, the fermentative product of glycolysis, reflects glycolytic fluxes and represses immune signaling activation decreasing inflammation in macrophages. Therefore, lactate levels reflect the metabolic status of the cells and has consequences for the inflammatory levels of the cells.

    In this study the authors show that 2-AA can affect the metabolic state of macrophages by increasing the glycolytic flux with the consequent increase in lactate levels and decrease in TCA flux. They also show that lactate decreases inflammation by suppressing 2-AA activation of NF-kBeta signaling and proinflammatory cytokine production.

    Using a murine model they show that addition of that 2-AA in mice infected with Pseudomonas aeruginosa results in an increase production of lactate and decrease of ATP in mice tissue, thus providing for 2-AA-mediated metabolic changes in vivo.

    The study described here is well written and the conclusions are generally well supported by the data. While they tested the direct effect of the 2-AA signal in macrophages, this was not tested in vivo in the absence of infection, and I think it is important to address the direct impact of the signal on the host.

    The study reported here proposes that a quorum sensing signal has an impact in pathogen persistence through immunometabolic reprograming properties, and provides evidence for a novel mechanism by which bacteria use quorum sensing signals to persist in the host.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    Tolerance in macrophages involves a global transcriptional shift from a pro-inflammatory response toward one characterized by the expression of anti-inflammatory and pro-resolution factors. In the case of TLR-mediated tolerance, pro-inflammatory cytokines are not universally suppressed in all tolerant cells, but distinct patterns of cytokine expression distinguished TLR-specific tolerance. (10.3389/fimmu.2018.00933, 10.1615/critrevimmunol.2015015495). However, the authors only show differences in TFNa. Thus, I strongly suggest the authors to determine anti-inflammatory cytokines, such as IL-10.

    Read the original source
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