The metabolic cofactor Coenzyme A enhances alternative macrophage activation via MyD88-linked signaling

  1. Anthony E. Jones
  2. Amy Rios
  3. Neira Ibrahimovic
  4. Carolina Chavez
  5. Nicholas A. Bayley
  6. Andréa B. Ball
  7. Wei Yuan Hsieh
  8. Alessandro Sammarco
  9. Amber R. Bianchi
  10. Angel A. Cortez
  11. Thomas G. Graeber
  12. Alexander Hoffmann
  13. Steven J. Bensinger
  14. Ajit S. Divakaruni

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Abstract

Metabolites and metabolic co-factors can shape the innate immune response, though the pathways by which these molecules adjust inflammation remain incompletely understood. Here we show that the metabolic cofactor Coenzyme A (CoA) enhances IL-4 driven alternative macrophage activation [m(IL-4)] in vitro and in vivo . Unexpectedly, we found that perturbations in intracellular CoA metabolism did not influence m(IL-4) differentiation. Rather, we discovered that exogenous CoA provides a weak TLR4 signal which primes macrophages for increased receptivity to IL-4 signals and resolution of inflammation via MyD88. Mechanistic studies revealed MyD88-linked signals prime for IL-4 responsiveness, in part, by reshaping chromatin accessibility to enhance transcription of IL-4-linked genes. The results identify CoA as a host metabolic co-factor that influences macrophage function through an extrinsic TLR4-dependent mechanism, and suggests that damage-associated molecular patterns (DAMPs) can prime macrophages for alternative activation and resolution of inflammation.

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    Reply to the reviewers

    Prior to the point-by-point response to the reviewer, we would like to sincerely thank all the peer reviewers for their overwhelmingly positive comments and helpful suggestions. The recommendations have undoubtedly improved our initial submission, and we have done our best to incorporate as many of the suggestions as possible.

    Reviewer #1* (Evidence, reproducibility and clarity (Required)): *

    *Jones et al. have submitted a manuscript detailing the role of Coenzyme A in the regulation of macrophage polarization. Overall, the manuscript is well designed, and the conclusions are well supported by the data. I find no major or minor deficiencies that need to be corrected. *

    Reviewer #1 (Significance (Required)): *

    For decades the immunology community has boldly stated that mitochondrial metabolism not only provides the bioenergetics for cell expansion but also dictates cell fate. This has been especially true for fatty acid beta oxidation. Macrophage, T-cell and B-cell polarization have all been shown to require FAO for their polarization, but all based on one inhibitor. NONE of these observations hold up with more rigorous experimentation. The Divakaruni group has previously suggested that intracellular CoA homeostasis was the driver of macrophage differentiation as they could reverse the inhibitory effects by providing heroic levels of CoA extracellularly. Here, they have clarified the role of CoA. Intracellular CoA does not affect macrophage polarization/differentiation. This was done with cleaver manipulation of the CoA pools. Rather, extracellular CoA can act as a weak TLR4 ligand. This work nicely clarifies their previous work and further demonstrates a role for this metabolite as an endogenous activator of type 1 macrophages.

    We are thrilled by the positive comments about our work, and we are grateful the reviewer found our submission to be clarifying for the field and significant in the larger context of immunometabolism research.

    Reviewer #2* (Evidence, reproducibility and clarity (Required)):*

    *This is a fairly straightforward manuscript that indicated CoA acts as a "weak" TLR4 agonist and primes macrophages for alternative activation. Overall, the experiments are well done and clear enough. There are two major issues that need to be addressed: *

    We thank the reviewer for their positive comments regarding the quality and clarity of our work.

    *Previous work has shown the following pathway: LPS>IL10>STAT3>IL4Ra>>>increased responsiveness to IL4/IL13 and increased expression of M2 associated markers (please note, this pathway does not apply to Arg1, often erroneously associated with M2 macrophages - LPS induces Arg1 far more than IL4 and this is independent of the STAT6 pathway - Dichtl et al., Science Advances and El Kasmi et al. Nature Immunology, and others). This pathway was first described in Lang et al. 2002 J. Immunol. Subsequently, other groups showed IL6 (Jens Brüning) and OSM (Carl Richards) do the same thing, which is not surprising given that they are STAT3 activators. Thus, Il4ra is a STAT3 target gene; this also makes sense in the kinetic evolution of macrophages from inflammatory to tissue reparative (if they survive). In my view, the authors have most likely found the same pathway. In Jones, expression of the IL4Ra was not quantified. Thus, the pathway described above needs to be accounted for. It may not apply here but seems the easiest explanation of the data. *

    This is an excellent and important experiment suggested by the reviewer, and we address this in our revised Supplemental Figure 5. To determine whether the effect of CoA can be explained simply by a STAT3-mediated effect on the IL-4 receptor, we treated cells with the well-characterized STAT3 inhibitor Napabucasin and measured whether CoA could enhance the macrophage IL-4 response. Two results are clear from the data:

    • Treatment with Napabucasin reduced the expression of IL-4-linked cell surface markers and the IL-4 target gene Ccl8. This serves as an important control consistent with the Il4ra gene being a STAT3 target that increases IL-4 responsiveness.
    • Despite STAT3 inhibition and a reduced IL-4 response, CoA provision still augmented the IL-4-induced expression of Ccl8 and the percentage of CD206+/CD301+ cells, indicating a STAT3-independent mechanism. The result aligns with our ATAC-Seq data in Figure 6 that shows broad changes in chromatin accessibility that cannot be completely explained by expression-level changes in the IL-4 receptor.

    *Can the authors come up with a meaningful in vivo experiment to corroborate their data. Pantothenate-deficient mice have many phenotypes (not fully explored at all - PMID 31918006, for example) and pantothenate metabolism can be manipulated in different ways. Obviously, a complex in vivo experiment is not feasible here. But this should be discussed. What happens in human macrophages, where "polarization" is a completely different beast? *

    We thank the reviewer for these thoughtful comments, and address the questions regarding in vivo proof-of-concept and polarization of human macrophages separately:

    • Regarding the question of whether CoA can enhance the phenotype of IL-4-activated human macrophages, this is an excellent suggestion and we have added the data as Figure 1h. Indeed, Coenzyme A dramatically amplifies expression of the human IL-4 responsive genes CCL17, TGM2, and PDCD1LG2 (similarly to mouse macrophages). The result substantially expands the significance of our work by showing the phenotype is reproducible in both mouse and human macrophages – unlike many immunometabolic phenotypes – and we thank the reviewer again for suggesting this experiment.
    • With respect to an in vivo experiment to corroborate our data, we entirely agree with the reviewer regarding both the importance, but also the difficulty in interpretation, of an experiment genetically manipulating CoA synthesis in vivo. As they have suggested, we raise these issues in the discussion on Lines 370-377 of the revised manuscript. Here, we note the following points:
    1. Wherever possible/appropriate (e.g. Figures 1g, 3f&g, 5g&h), we have sought to corroborate our in vitro findings with in vivo/ex vivo proofs-of-concept.
    2. Studying immune phenotypes in pantothenate-deficient mice would be an exciting experiment in principle, but difficult to interpret if conducted. As noted by the reviewer in the work from Drs. Rock and Jackowski, knockout of one of four isoforms of pantothenate kinase (PANK) shows mild phenotypes consistent with compensation across isoforms for CoA provision. Global double knockout of PANK1 and PANK2, however, is postnatally lethal. Regardless, a tissue-specific double knockout in myeloid cells is unlikely to show a phenotype given our results showing that manipulating intracellular CoA levels in BMDMs does not alter the IL-4 response (Figs. 2h-j).
    3. Given the established role of CoA in postnatal development, it would be difficult to attribute any immunologic phenotypes in genetically modified mice to direct effects of CoA as a metabolic DAMP as opposed to indirect effects from a chronically altered immune system.

    Reviewer #2 (Significance (Required)): *This is a fairly straightforward manuscript that indicated CoA acts as a "weak" TLR4 agonist and primes macrophages for alternative activation. Overall, the experiments are well done and clear enough.

    We reiterate our gratitude for the comments on the quality and clarity of our work.

    Reviewer #3* (Evidence, reproducibility and clarity (Required)):*

    Summary: In this manuscript on enhancement of mIL-4 polarization by exogenous CoA, the authors follow up on their previous studies that had shown a correlation between Etomoxir-driven block in mIL-4 and a reduction of intracellular CoA levels. The results obtained (lack of enhancement of IL-4-induced changes in oxidative phosphorylation and glycolysis; lack of impact of pharmacological decrease/increase of intracellular CoA levels) led them to discard their initial hypothesis. Instead, the presence of a proinflammatory gene signature in macrophages treated with IL-4+CoA triggered experiments testing the involvement of TLR-Myd88 signaling and the identification of CoA as a weak agonist for TLR4 (which is consistent with a preprint manuscript posted in 2022 by others and showing induction of proinflammatory gene express in a TLR2/4-dependent manner).

    Significance: Overall, these results are novel and interesting, although the use of yeast-derived CoA preparations raises a question about the contribution of contaminants that is only partially controlled by data obtained with a synthetic CoA. Regarding a biological role for CoA in macrophage biology in vivo, the authors propose that CoA may act as a DAMP upon release from dying/dead cells and thereby modify transcriptional polarization of m(IL-4). I have several comments related to specific experimental conditions and interpretation that should be addressed. Most importantly, the key findings of the manuscript should be demonstrated using synthetic CoA as described in comment #5.

    We are heartened that the reviewer found our initial submission to be novel and interesting, and are grateful for their suggestions to reinforce our existing data with more studies comparing yeast-derived and synthetically-derived Coenzyme A. We have done our best to address each of the individual questions below:

    Major comments:

    *Increasing/decreasing intracellular CoA levels does not alter IL-4-induced CD206 expression (Fig. 2i/j. However, the impact of CoA addition to mIL-4 is stronger for Ccl8 and Mgl2 mRNA (Fig. 1a) than for the CD206+ cell fraction (Fig. 1d). Therefore, it would be better (higher sensitivity) to include expression of these genes as readout after CPCA/PZ-2891 treatment. *

    This is a helpful suggestion, and we have now conducted gene expression studies to complement our flow cytometry and mass spectrometry studies while manipulating the intracellular CoA pool. In line with our previous work, neither CPCA (which decreases intracellular free CoA) or PZ-2891 (which increases intracellular free CoA) meaningfully alter expression of IL-4-linked genes including Ccl8 or Mgl2. In fact, the only (statistically insignificant) trend refutes the hypothesis, as gene expression with CPCA leads to marginally increased gene expression. These results are now included in Supplemental Figure S2f. We thank the reviewer for this helpful suggestion, as it has strengthened our conclusion that intracellular CoA levels do not adjust the macrophage IL-4 response.

    • The CoA-induced proinflammatory gene expression in Fig. 3c is relatively weak (e.g. compared to LPS). The authors use CoA throughout the manuscript at a concentration of 1 mM, and we do not know how much of it is required to cause an effect at all. Therefore, dose-response curves for the stimulation of macrophages with titrated amounts of CoA should be provided. In addition, *

    We thank the reviewer for bringing up this point so we could clarify and add to our existing data. We should note that Supplemental Figures 1b&c of our previous submission (and resubmitted manuscript) detail a concentration-response curve showing that at little as 62.5 mM CoA – the lowest concentration tested – was sufficient to enhance IL-4 cell surface marker expression.

    However, it is an excellent suggestion as the reviewer notes, to conduct a similar concentration-response to determine if this lines up with CoA inducing a pro-inflammatory response. The full data set is presented in the answer to reviewer question 4 (comparing CoA purchased from Sigma vs. Avanti Polar Lipids), though we now show in Supplemental Figure S3 that 62.5 mM CoA is sufficient to elicit a pro-inflammatory response. Though it is indeed a weak effect as noted by the reviewer, our data suggest that the relatively mild stimulus is crucial for the effect. Given the results with the TLR3 agonist Poly I:C (Figure 5), which engages a Type 1 interferon response, strong TLR4 agonists that engage the TRIF/Type I interferon arm of the TLR4 response are likely to blunt or block the IL-4 response.

    • Related question: we are informed that the concentration of CoA in the mitochondrial matrix is 5mM, whereas cytosol contains 100µM. For CoA to act as DAMP, I would like to know the concentration of it in supernatants of cell cultures (live vs. dying/dead cells) and from tissues. *

    This is an important point brought up by the reviewer, and we agree that the implicit issue raised (i.e. “do the concentrations of CoA required to see an effect reconcile with a physiological role as a DAMP?”) should be more thoroughly addressed in the manuscript. Tissue concentrations of free CoA (in ng/mg tissue) are well established for mice and range from >100 nmol/g tissue (liver, heart, brown adipose tissue) to Nonetheless, the reviewer’s larger point is very well reasoned, and we address it in the following ways in the discussion on __Lines 378-391. __

    • In light of the reviewer’s comment, we now mention specific instances in the discussion where CoA acting as a DAMP may reasonably play a physiological role (e.g. acetaminophen-induced acute liver injury or other forms of sterile liver injury given that DAMPs are known to be important factors and liver tissue contains relatively high concentrations of CoA).
    • Although cytoplasmic concentrations of CoA may only be 50-100 mM, our work establishes a framework for how ubiquitous metabolic co-factors can activate pattern recognition receptors. Put another way, although CoA itself may not be a physiologically relevant DAMP, discovering this pathway could inform how other nucleotide or nucleoside analogs (e.g. adenine- or adenosine-containing molecules present at millimolar concentrations) exert their effects on innate immunity.
    • Our newly obtained data with HMDMs (Figure 1h) shows that the CoA response in human macrophages – boosting IL-4-linked gene expression by 10-100X – may be much stronger than the 1.5-5X effect observed in mouse BMDMs. As such, it is exciting to speculate that CoA may have a more potent effect on the IL-4 response in humans relative to mice. We trust the reviewer understands the limitations of obtaining human macrophages that preclude conducting a thorough concentration-response analysis given the restrictions of a manuscript revision.
    • It is very good that the authors validate the findings obtained using the yeast-derived CoA with the synthetic molecule. It is very conceivable that the 15% contaminating substances in the yeast CoA could be causing the observed changes in m(IL-4). The fact that synthetic CoA has higher activity in proinflammatory gene expression by BMM (Suppl. Fig. S3) is reassuring, however, it raises the question why this is the case. One possibility is that the concentrations of the different CoA preps cannot directly be compared. Therefore, dose response curves should also be provided for synthetic CoA. *

    This is an astute observation by the reviewer and we thank them for reading our manuscript with such detailed attention to pick this up. We are reassured that the reviewer shares our interpretation that the effect of CoA is not due to a contaminating TLR4 agonist in the yeast-derived preparation (from Sigma-Aldrich; ~85% pure) given a negative Limulus Test (Supplemental Figure S4b). Moreover, the synthetically-derived preparation (from Avanti Polar Lipids; ~99% pure) yields a stronger TLR4 response.

    An exploration of the follow-on question regarding why the effect is greater than 15% is presented below. These experiments have been added to Supplemental Figure S4c&d. The summary of our data suggests the individual concentrations indeed cannot be compared – matched concentrations of synthetic Avanti CoA have greater than a 15% effect than yeast-derived Sigma CoA. There are likely multiple factors that could explain this, some of which are listed below.

    • The physiological effect of a TLR agonist need not be linear with its concentration, as demonstrated by the sigmoidal calibration curves for the TLR-expressing HEK-blue cells (Figures 4b, S4a). This likely does not explain the dramatic difference between the two CoA preparations but is worth noting.
    • While we have determined that the 15% contaminating substances in the yeast-derived CoA are not causing the observed changes in the IL-4 response, it is formally possible that there are contaminating substances blunting the pro-inflammatory response and therefore limiting the effect of CoA purchased from Sigma-Aldrich relative to that from Avanti Polar Lipids. Importantly, however, our data in response to Reviewer Question #5 show there is no difference in amplifying the IL-4 response between the yeast- and synthetically-derived CoA.
    • The difference in activity of yeast and synth. CoA could also be caused by the additional biologically active molecules in the yeast CoA. Therefore, it is important to show that the key findings in the paper (enhancement of m(IL-4) associated gene expression and CD206+ upregulation in vitro and in vivo) are also induced by synth. CoA. This is even more important in the context of the Myd88-independence of CD206+ upregulation in BMM treated with CoA (Suppl. Fig. S4). The experiment should be repeated with synth. CoA. If the enhancement of CD206+ cells induced by CoA is indeed unchanged in Myd88 KO BMM, then the title of the manuscript "CoA enhances alternative macrophage activation via Myd88" would not be supported by the data and needed to be changed. Activation of the TLR4 reporter cell line should also be tested using the synth. CoA molecule.*

    We are grateful for this suggestion by the reviewer to further cement the idea that our observation of CoA enhancing the macrophage IL-4 response was not due to a contaminant in the Sigma-Aldrich CoA preparation. The reviewer makes a few points in this question which we address individually here.

    • The suggestion to confirm that the CoA-induced enhancement of M(IL-4) is not due to a contaminating substance in the Sigma-Aldrich CoA is excellent and necessary. Here we show that synthetically derived CoA (99% pure, purchased from Avanti Polar Lipids) quantitatively reproduces the effect from yeast-derived CoA from Sigma-Aldrich in Supplemental Figure S4e. The response is noteworthy because synthetic CoA has profoundly stronger pro-inflammatory response than yeast-derived CoA, yet both have a similar effect on augmenting M(IL-4). This suggests that any appropriate pro-inflammatory response – irrespective of the relative strength or weakness – is sufficient to maximize the effect. This can also be observed with the range of MyD88-linked TLR agonists used in Figures 5 and S6a.
    • Similarly, we also conducted experiments to show that the effect of synthetic CoA on M(IL-4) is independent of MyD88 similarly to yeast-derived CoA. These data are present in Supplemental Figure S6b&c. Here again, we should note that the effect of synthetic CoA is quantitatively similar to the effect of yeast CoA and Imiquimod (Supplemental Figure S6a).
    • Activation of the TLR4 reporter cell line is available in Supplemental Figure S4c.
    • Regarding the title of the manuscript, we acknowledge that we struggled a bit with how to frame our findings. Importantly, our findings support a model where (i) CoA provision enhances the IL-4 response not via metabolic changes but rather by acting as a mild pro-inflammatory stimulus, and (ii) MyD88 signaling augments the IL-4 response. We should also note that our findings simply show that CoA does not exclusively enhance the IL-4 response via MyD88 signaling, and there may be other redundant pathways (similarly to MyD88 agonist imiquimod but unlike the MyD88 agonists Pam3-CSK4 and low concentrations of LPS). We are open to working journal editors to strike the right balance of scientific accuracy and representation of the work when deciding on a final title.
    • The results from the tumor model in Fig. 5 are presented to show a stronger tumor-promoting effect of m(IL-4) stimulated with Pam3. However, the variability of the data is high and 2 out of 6 mice in the +Pam3 group appear to actually have a lower tumor weight than the control mice. Therefore, these data are quite superficial and preliminary, and would benefit from a replicate experiment. Furthermore, for the evaluation of CoA as a biologically relevant DAMP, it would be important to know whether CoA-treated m(IL-4) show the same tumor-promoting effect in vivo as Pam3. *

    We thank the reviewer for their comment, and agree that our in vivo work is indeed preliminary. Our goal with this report was to focus on the initial discovery of this molecular pathway and its first, broad characterization using a range of techniques (e.g. in vivo outcomes, ATAC-Seq, etc.), many of which can spur more detailed follow-up studies for future papers. As detailed in the manuscript discussion (Lines 415-419), future work beyond our initial discovery is warranted to thoroughly explore the physiological outcomes of CoA as a metabolic DAMP in relevant model systems such as acute liver injury. As an initial proof-of-concept to show that MyD88 signaling can enhance alternative activation, however, we believe our two discrete experiments (sterile inflammation and tumor formation) are sufficient to indicate the phenotype is likely relevant in animal models. In vivo syngeneic tumor models display natural variability in tumor size due to differences in implantation efficiency, host immune responses, and tumor-intrinsic growth kinetics. Nonetheless, our statistical analysis demonstrates that, with high confidence, that the observed differences are reproducible and not attributable to random variation.

    Minor comments:

    • Fig. 1b: where the gates for CD206/CD301 set based on isotype control stainings? *

    We thank the reviewer for pointing out this oversight in our methods. The gates were indeed set on isotype control stains, and this is now mentioned in Lines 519-521 of the revised manuscript.

    The formatting not cohesive m(IL-4) vs. M(IL-4)

    Again, this is an embarrassing oversight on our part and we have done our very best to copy edit the piece and remove any inconsistencies and errors.

    *Methods: primer sequences are not shown. They should be provided. *

    We thank the reviewer for pointing this out, and now include all primer sequences used in Supplemental Table 1 of the revised manuscript.

    Description of flowcytometry (L/D staining after surface? No washing steps after addition of L/D staining)

    We thank the reviewer for pointing out another oversight in our methods, and have provided a more detailed description of the flow cytometric analysis in Lines 509-521 of the revised manuscript.

    Statistics: the methods section states that variability is indicated by SD, but the Figure legends always mention SEM. Please correct.

    We are grateful for the reviewer’s helpful attention to detail, and have corrected the methods to line up with the figure legends.

    *A multitude of typos and editorial inconsistencies (e.g. spelling of m(IL-4), punctation and capitalization) should be corrected/streamlined. *

    We are grateful for the reviewer’s helpful attention to detail, and have done our best to copy edit the manuscript prior to resubmission.

    Reviewer #3 (Significance (Required)):

    strengths: I like that the authors follow up their previous work on Etomoxir and CoA, now finding again an unexpected twist in how the effect on m(IL-4) is brought about. This makes the story more complicated, but is important to get to a more precise and realistic understanding of metabolic and transcriptomic regulation and how they are interconnected (or not). In addition, the use of a relatively broad set of methods including ATACseq and mass spectrometry is a strength.

    weakness: the use of the not very pure yeast derived CoA prep, which is controlled for induction of proinflammatory cytokines by one experiment with synth. CoA. This validation needs to be expanded (see comments above) to substantiate the main message of the manuscript.

    The scope of the manuscript is quite focussed on the mechanism of CoA enhanced m(IL-4). The finding that CoA appears not to act by changing intracellular macrophage metabolism but instead after its release by activation TLR4 widens the scope and suggests a new function for CoA as DAMP. This aspect would need to be further substantiated to be convincing.

    Audience: scientists working at the intersection between metabolism and innate immunity will be interested in the results.

    We thank the reviewer for their kind comments regarding the precision, credibility, and breadth of our manuscript. We hope they find our revised manuscript an improvement over our previous submission regarding both the new experiments and modified text. The comments have undoubtedly improved our manuscript and we are grateful to the reviewer for the considerable effort they put into reading our submission.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript on enhancement of mIL-4 polarization by exogenous CoA, the authors follow up on their previous studies that had shown a correlation between Etomoxir-driven block in mIL-4 and a reduction of intracellular CoA levels. The results obtained (lack of enhancement of IL-4-induced changes in oxidative phosphorylation and glycolysis; lack of impact of pharmacological decrease/increase of intracellular CoA levels) led them to discard their initial hypothesis. Instead, the presence of a proinflammatory gene signature in macrophages treated with IL-4+CoA triggered experiments testing the involvement of TLR-Myd88 signaling and the identification of CoA as a weak agonist for TLR4 (which is consistent with a preprint manuscript posted in 2022 by others and showing induction of proinflammatory gene express in a TLR2/4-dependent manner).

    Significance:

    Overall, these results are novel and interesting, although the use of yeast-derived CoA preparations raises a question about the contribution of contaminants that is only partially controlled by data obtained with a synthetic CoA. Regarding a biological role for CoA in macrophage biology in vivo, the authors propose that CoA may act as a DAMP upon release from dying/dead cells and thereby modify transcriptional polarization of m(IL-4). I have several comments related to specific experimental conditions and interpretation that should be addressed. Most importantly, the key findings of the manuscript should be demonstrated using synthetic CoA as described in comment #5.

    Major comments:

    1. Increasing/decreasing intracellular CoA levels does not alter IL-4-induced CD206 expression (Fig. 2i/j. However, the impact of CoA addition to mIL-4 is stronger for Ccl8 and Mgl2 mRNA (Fig. 1a) than for the CD206+ cell fraction (Fig. 1d). Therefore, it would be better (higher sensitivity) to include expression of these genes as readout after CPCA/PZ-2891 treatment.
    2. The CoA-induced proinflammatory gene expression in Fig. 3c is relatively weak (e.g. compared to LPS). The authors use CoA throughout the manuscript at a concentration of 1 mM, and we do not know how much of it is required to cause an effect at all. Therefore, dose-response curves for the stimulation of macrophages with titrated amounts of CoA should be provided. In addition,
    3. Related question: we are informed that the concentration of CoA in the mitochondrial matrix is 5mM, whereas cytosol contains 100µM. For CoA to act as DAMP, I would like to know the concentration of it in supernatants of cell cultures (live vs. dying/dead cells) and from tissues.
    4. It is very good that the authors validate the findings obtained using the yeast-derived CoA with the synthetic molecule. It is very conceivable that the 15% contaminating substances in the yeast CoA could be causing the observed changes in m(IL-4). The fact that synthetic CoA has higher activity in proinflammatory gene expression by BMM (Suppl. Fig. S3) is reassuring, however, it raises the question why this is the case. One possibility is that the concentrations of the different CoA preps cannot directly be compared. Therefore, dose response curves should also be provided for synthetic CoA.
    5. The difference in activity of yeast and synth. CoA could also be caused by the additional biologically active molecules in the yeast CoA. Therefore, it is important to show that the key findings in the paper (enhancement of m(IL-4) associated gene expression and CD206+ upregulation in vitro and in vivo) are also induced by synth. CoA. This is even more important in the context of the Myd88-independence of CD206+ upregulation in BMM treated with CoA (Suppl. Fig. S4). The experiment should be repeated with synth. CoA. If the enhancement of CD206+ cells induced by CoA is indeed unchanged in Myd88 KO BMM, then the title of the manuscript "CoA enhances alternative macrophage activation via Myd88" would not be supported by the data and needed to be changed. Activation of the TLR4 reporter cell line should also be tested using the synth. CoA molecule.
    6. The results from the tumor model in Fig. 5 are presented to show a stronger tumor-promoting effect of m(IL-4) stimulated with Pam3. However, the variability of the data is high and 2 out of 6 mice in the +Pam3 group appear to actually have a lower tumor weight than the control mice. Therefore, these data are quite superficial and preliminary, and would benefit from a replicate experiment. Furthermore, for the evaluation of CoA as a biologically relevant DAMP, it would be important to know whether CoA-treated m(IL-4) show the same tumor-promoting effect in vivo as Pam3.

    Minor comments:

    1. Fig. 1b: where the gates for CD206/CD301 set based on isotype control stainings?
    2. The formatting not cohesive m(IL-4) vs. M(IL-4)
    3. Methods: primer sequences are not shown. They should be provided.
    4. Description of flowcytometry (L/D staining after surface? No washing steps after addition of L/D staining)
    5. Statistics: the methods section states that variability is indicated by SD, but the Figure legends always mention SEM. Please correct.
    6. A multitude of typos and editorial inconsistencies (e.g. spelling of m(IL-4), punctation and capitalization) should be corrected/streamlined.

    Significance

    Strengths: I like that the authors follow up their previous work on Etomoxir and CoA, now finding again an unexpected twist in how the effect on m(IL-4) is brought about. This makes the story more complicated, but is important to get to a more precise and realistic understanding of metabolic and transcriptomic regulation and how they are interconnected (or not). In addition, the use of a relatively broad set of methods including ATACseq and mass spectrometry is a strength.

    Weakness: the use of the not very pure yeast derived CoA prep, which is controlled for induction of proinflammatory cytokines by one experiment with synth. CoA. This validation needs to be expanded (see comments above) to substantiate the main message of the manuscript.

    The scope of the manuscript is quite focussed on the mechanism of CoA enhanced m(IL-4). The finding that CoA appears not to act by changing intracellular macrophage metabolism but instead after its release by activation TLR4 widens the scope and suggests a new function for CoA as DAMP. This aspect would need to be further substantiated to be convincing.

    Audience: scientists working at the intersection between metabolism and innate immunity will be interested in the results.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This is a fairly straightforward manuscript that indicated CoA acts as a "weak" TLR4 agonist and primes macrophages for alternative activation. Overall, the experiments are well done and clear enough. There are two major issues that need to be addressed:

    1. Previous work has shown the following pathway: LPS>IL10>STAT3>IL4Ra>>>increased responsiveness to IL4/IL13 and increased expression of M2 associated markers (please note, this pathway does not apply to Arg1, often erroneously associated with M2 macrophages - LPS induces Arg1 far more than IL4 and this is independent of the STAT6 pathway - Dichtl et al., Science Advances and El Kasmi et al. Nature Immunology, and others). This pathway was first described in Lang et al. 2002 J. Immunol. Subsequently, other groups showed IL6 (Jens Brüning) and OSM (Carl Richards) do the same thing, which is not surprising given that they are STAT3 activators. Thus, Il4ra is a STAT3 target gene; this also makes sense in the kinetic evolution of macrophages from inflammatory to tissue reparative (if they survive). In my view, the authors have most likely found the same pathway. In Jones, expression of the IL4Ra was not quantified. Thus, the pathway described above needs to be accounted for. It may not apply here but seems the easiest explanation of the data.
    2. Can the authors come up with a meaningful in vivo experiment to corroborate their data. Pantothenate-deficient mice have many phenotypes (not fully explored at all - PMID 31918006, for example) and pantothenate metabolism can be manipulated in different ways. Obviously, a complex in vivo experiment is not feasible here. But this should be discussed. What happens in human macrophages, where "polarization" is a completely different beast?

    Significance

    This is a fairly straightforward manuscript that indicated CoA acts as a "weak" TLR4 agonist and primes macrophages for alternative activation. Overall, the experiments are well done and clear enough.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Jones et al. have submitted a manuscript detailing the role of Coenzyme A in the regulation of macrophage polarization. Overall, the manuscript is well designed, and the conclusions are well supported by the data. I find no major or minor deficiencies that need to be corrected.

    Significance

    For decades the immunology community has boldly stated that mitochondrial metabolism not only provides the bioenergetics for cell expansion but also dictates cell fate. This has been especially true for fatty acid beta oxidation. Macrophage, T-cell and B-cell polarization have all been shown to require FAO for their polarization, but all based on one inhibitor. NONE of these observations hold up with more rigorous experimentation. The Divakaruni group has previously suggested that intracellular CoA homeostasis was the driver of macrophage differentiation as they could reverse the inhibitory effects by providing heroic levels of CoA extracellularly. Here, they have clarified the role of CoA. Intracellular CoA does not affect macrophage polarization/differentiation. This was done with cleaver manipulation of the CoA pools. Rather, extracellular CoA can act as a weak TLR4 ligand. This work nicely clarifies their previous work and further demonstrates a role for this metabolite as an endogenous activator of type 1 macrophages.

  5. Version published to 10.1101/2024.03.28.587096 on bioRxiv