ATP-Evoked Intracellular Ca2+ Responses in M-CSF Differentiated Human Monocyte-Derived Macrophage are Mediated by P2X4 and P2Y11 Receptor Activation
Round 1
Reviewer 1 Report
In this study, the authors investigate ATP-induced calcium signals in human monocyte-derived macrophages and provide pharmacological demonstration of the purinergic receptors involved.
Most of the experiments are nicely performed and described. However, the study has limited novelty, as the presence of ATP-induced calcium signals in macrophages has already been reported in the literature (for example here: https://www.pnas.org/content/101/25/9479).
MAJOR POINTS:
The authors focus the attention on the specificity of ATP-induced calcium signals in M-CSF-differentiated macrophages, with respect to GM-CSF macrophages; however, the two macrophage populations are compared only in figure 1E and figure 3. It would be important to compare the expression of purinergic receptors in the two macrophage populations not only at the mRNA level, but also at the protein level, for example by western blot. (Data on the expression of purinergic receptors in GM-CSF macrophages have been reported by the same authors elsewhere, but they cannot be compared here). A very important point of the study is the identification of the purinergic receptors responsible for calcium signals; however, this result is supported only by pharmacological inhibition. Have the authors tried to downregulate the receptors’ expression? The authors should test the effect of the combination of P2X4 and P2Y11 antagonists on ATP-induced calcium signals. The authors should test whether P2X4 and/or P2Y11 play relevant roles on the cellular function of M-CSF-differentiated macrophages.MINOR POINTS:
The authors should improve the introduction of the article, as the cited literature is not very recent. In particular, the distinction M1 vs M2 macrophages should be discussed in more detail. The material and methods section lacks a paragraph illustrating how statistical analysis were performed. The same information is missing from the figure legends (statistical test employed, error bars). The commercial source of many of the reagents, for example the cytokines and the purinergic receptor antagonists, are not indicated. Also the treatment duration should be indicated. Despite the expression of P2Y4 at the mRNA level is detectable in M-CSF-differentiated macrophages, no data on the expression of the protein (fig. 4) or P2Y4 inhibition (fig. 5) is reported. The authors should explain why this receptor was excluded by the analysis.Author Response
In this study, the authors investigate ATP-induced calcium signals in human monocyte-derived macrophages and provide pharmacological demonstration of the purinergic receptors involved.
Most of the experiments are nicely performed and described. However, the study has limited novelty, as the presence of ATP-induced calcium signals in macrophages has already been reported in the literature (for example here: https://www.pnas.org/content/101/25/9479).
RESPONSE: Thank you. We are aware of this publication however our manuscript differs substantially. In the paper by Hanley et al., 2004 no attempt is made to use selective receptor antagonists to dissect the ATP-evoked Ca2+ response. Nor is any attempt made to confirm the expression of receptor subtypes at the protein level.
MAJOR POINTS:
The authors focus the attention on the specificity of ATP-induced calcium signals in M-CSF-differentiated macrophages, with respect to GM-CSF macrophages; however, the two macrophage populations are compared only in figure 1E and figure 3. It would be important to compare the expression of purinergic receptors in the two macrophage populations not only at the mRNA level, but also at the protein level, for example by western blot. (Data on the expression of purinergic receptors in GM-CSF macrophages have been reported by the same authors elsewhere, but they cannot be compared here).
RESPONSE: Expression at the protein level has been confirmed by immunocytochemistry (Figure 4). We do not see the value in comparing the protein level of each receptor subtype but have instead used intracellular Ca2+ measurements to make a functional comparison. Western blotting would not determine the quantity of functional receptor available to respond to ATP at the plasma membrane.
A very important point of the study is the identification of the purinergic receptors responsible for calcium signals; however, this result is supported only by pharmacological inhibition. Have the authors tried to downregulate the receptors’ expression? The authors should test the effect of the combination of P2X4 and P2Y11 antagonists on ATP-induced calcium signals. The authors should test whether P2X4 and/or P2Y11 play relevant roles on the cellular function of M-CSF-differentiated macrophages.
RESPONSE: Thank you for these experimental suggestions. We have attended to knockout P2 receptor subtypes in these cells using both siRNA and lentivirus-mediated shRNA delivery. Due to the primary nature of the cells used, both these methods have been unsuccessful in our hands. We have identified the relevant roles of P2X4 and P2Y11 in terms of intracellular Ca2+ response. The Editor has requested a response to reviewers within 10 days and therefore further experimentation has not been possible.
MINOR POINTS:
The authors should improve the introduction of the article, as the cited literature is not very recent. In particular, the distinction M1 vs M2 macrophages should be discussed in more detail.
RESPONSE: We have now improved the introduction regarding M1 vs M2 macrophage including recent literature.
The material and methods section lacks a paragraph illustrating how statistical analysis were performed. The same information is missing from the figure legends (statistical test employed, error bars).
RESPONSE: Thank you for this suggestion, and you are quite right that this should have been included – apologies. To this end, the following a statement has been included in the methods section. In addition, the statistical test employed and definition of errors bars as SEM now appears in relevant figure legends.
The commercial source of many of the reagents, for example the cytokines and the purinergic receptor antagonists, are not indicated. Also the treatment duration should be indicated.
RESPONSE: Information on cytokines is now included in the methods. Also a statement regarding the source of purinergic antagonist and application time is included.
Despite the expression of P2Y4 at the mRNA level is detectable in M-CSF-differentiated macrophages, no data on the expression of the protein (fig. 4) or P2Y4 inhibition (fig. 5) is reported. The authors should explain why this receptor was excluded by the analysis.
RESPONSE: Selective P2Y4 receptor antagonists are not commercially available and therefore the contribution of P2Y4 to the ATP-evoked Ca2+ response could not be tested. A commercially available and validated anti-P2Y4 antibody was not available at the time of experimentation.
Reviewer 2 Report
Comments on MS IJMS – 582732 “ATP-evoked intracellular Ca2+ responses in M-CSF differentiated human monocyte-dervived macrophage are mediated by P2X4 and P2Y11 receptor activation
The authors described the role of P2X4 and P2Y11 in the ATP-induced Ca2+ mobilization in macrophages.
Despite a very interesting work, this MS presents some weaknesses which impair its publication in this state
Major concerns:
It is well admitted that the main Ca2+ mobilization pathways in macrophages and leukocytes in general is due to the couple Orai1/STIM1 proteins. In their work the authors clearly established the role of P2Y11R in Ca2+ mobilization, acting more than probably through the IP3 pathways, leading to a Ca2+ entry through Orai1 channels. But there is not even a single sentence about this so important pathway in the introduction or in the discussion. U73122 inhibits the Ca2+ mobilization by 75%, meaning ¾ of the cytosolic Ca2+ come from the reticulum +/- the store-operated calcium entry through Orai1 channels. The authors should talk about it, and it did not need new experiments.
The kinetics of Ca2+ mobilization and its analysis. In discussion, line 200, the authors wrote about a “biphasic intracellular response consisting of a rapid peak phase, followed by a slower plateau phase”. I agree with the peak phase, but not with the plateau phase. For example, just above this text, in figure 7, especially fig 7A, there is only a peak followed by something looking like an inactivation with a return to the basic level, so where is the plateau ? Only in fig 2B, there is no fast return to the basic level, and a very tiny plateau exists.
3.About the kinetics analysis, it is a bit surprising that in some figure, the decay is simple and seems exponential, when in some other figures, a clear “bump” exists like in fig 6B. Can the authors wrote about this difference in kinetics ? Do they have any idea of what happens ?
P2X7R. All the P2X7R specialists will be astonished by how the authors study its role here. 100 µM ATP is not enough to activated P2X7R… So, it is normal that its specific inhibitor A438079 has no effect. To assess P2X7R role, the authors should repeat this kind of experiments, but after stimulation with 500 µM, or 10 µM Bz-ATP. It is interesting to have a look on fig 2A, where 100 µM ATP does not fully activated the Ca2+ mobilization, you need more… maybe due to P2X7R…
it is now very common after a pharmacological study highlighting the role of a receptor to use si or shRNA to impair its expression. Did the authors try to decrease P2X4 and P2Y11 expression by this way ? An alternative could be to isolate monocytes form already existing P2X4 KO mice.
Minor concerns:
line 118, fig 2B&C “25% of the response was resistant to U73122 inhibition”. From the example of fig 2B, it cannot be 25%, but less if the area undercurve was calculated, but seems exact if the % Ca2+ peak. So what was the calculation ?Many P2X and P2Y were expressed by the cells, but the use of specific inhibitors shows only a role for P2X4 and P2Y11… can you discuss why the others have no role ?
Ivermectin is not so easy to use. It is ok if you do patch-clamping on P2X4R-expressing cells, but trickier with other technics. Thus, Ivermectin is a well known inhibitor of SERCA(Ahern 1999), meaning an increase of Ca2+ concentration could be due to a potentiation of P2X4R, but also to a decreased Ca2+ pumping activity of the reticulum.
What about the fact that some inhibitors increased the Ca2+ mobilization ? That is the case for P2Y2 and P2X1.
Author Response
It is well admitted that the main Ca2+ mobilization pathways in macrophages and leukocytes in general is due to the couple Orai1/STIM1 proteins. In their work the authors clearly established the role of P2Y11R in Ca2+ mobilization, acting more than probably through the IP3 pathways, leading to a Ca2+ entry through Orai1 channels. But there is not even a single sentence about this so important pathway in the introduction or in the discussion.
RESPONSE: Thank you for this important point. The following statement will be included in the discussion “…the majority of the magnitude of the ATP-evoked Ca2+ response was due to activation of metabotropic P2Y receptors, revealed by U73122 sensitivity. Though not tested here, coupling of Ca2+ mobilisation to Orai1/STIM1 proteins are likely to be involved also as they have been shown to be important in GPCR-mediated Ca2+ signals in macrophage and microglia (Ferreira & Schlichter, 2013; Vaeth et al., 2015).
U73122 inhibits the Ca2+ mobilization by 75%, meaning ¾ of the cytosolic Ca2+ come from the reticulum +/- the store-operated calcium entry through Orai1 channels. The authors should talk about it, and it did not need new experiments.
RESPONSE: We do not agree with this interpretation of the data. Our interpretation is that in the presence of Ca2+ metabotropic responses (P2Y) contribution 75% at maximum ATP concentrations and 25% from activation of P2X receptors (direct Ca2+ influx). This is discussed.
The kinetics of Ca2+ mobilization and its analysis. In discussion, line 200, the authors wrote about a “biphasic intracellular response consisting of a rapid peak phase, followed by a slower plateau phase”. I agree with the peak phase, but not with the plateau phase. For example, just above this text, in figure 7, especially fig 7A, there is only a peak followed by something looking like an inactivation with a return to the basic level, so where is the plateau ? Only in fig 2B, there is no fast return to the basic level, and a very tiny plateau exists.
RESPONSE: Thank you for this suggestion. We agree that the description of a plateau phase is not accurate and will we remove this description.
3.About the kinetics analysis, it is a bit surprising that in some figure, the decay is simple and seems exponential, when in some other figures, a clear “bump” exists like in fig 6B. Can the authors wrote about this difference in kinetics ? Do they have any idea of what happens ?
RESPONSE: We attribute this to donor-to-donor variability of cells as it’s occurrence was random.
P2X7R. All the P2X7R specialists will be astonished by how the authors study its role here. 100 µM ATP is not enough to activated P2X7R… So, it is normal that its specific inhibitor A438079 has no effect. To assess P2X7R role, the authors should repeat this kind of experiments, but after stimulation with 500 µM, or 10 µM Bz-ATP. It is interesting to have a look on fig 2A, where 100 µM ATP does not fully activated the Ca2+ mobilization, you need more… maybe due to P2X7R…
RESPONSE: We are aware that we can use very high concentrations of ATP, use non-physiological divalent cations conditions, or use artificial agonists such as bzATP to isolate a P2X7-specific response. However, this study focuses on the natural ligand ATP under physiologically-relevant conditions. We use 100uM in the study as this is the maximum point on the ATP concentration response curve (Figure 2A; not significantly different from 300uM or 1mM ATP response). We are not denying that P2X7 is expressed by macrophage, indeed we demonstrate its expression in Figure 4, but under these conditions P2X7 does not contribute to the ATP-evoked Ca2+ response.
The following discussion point is included in the original manuscript:
“In keeping with our previous observations in human primary macrophage [10], P2X7 does not contribute to the Ca2+response at maximal ATP contributions. Others have observed P2X7 receptor contribution with the agonist BzATP, or at supramaximal ATP concentrations in the presence of unphysiologically low divalent cation concentrations [22-24]”
It is now very common after a pharmacological study highlighting the role of a receptor to use si or shRNA to impair its expression. Did the authors try to decrease P2X4 and P2Y11 expression by this way ? An alternative could be to isolate monocytes form already existing P2X4 KO mice.
RESPONSE: Thank you for these experimental suggestions. We have attended to knockout P2 receptor subtypes in these cells using both siRNA and lentivirus-mediated shRNA delivery. Due to the primary nature of the cells used, both these methods have been unsuccessful in our hands. We have identified the relevant roles of P2X4 and P2Y11 in terms of intracellular Ca2+ response. The Editor has requested a response to reviewers within 10 days and therefore further experimentation has not been possible.
As the study focused on human macrophage, work with mice would not be mechanistically insightful.
Minor concerns:
line 118, fig 2B&C “25% of the response was resistant to U73122 inhibition”. From the example of fig 2B, it cannot be 25%, but less if the area undercurve was calculated, but seems exact if the % Ca2+ peak. So what was the calculation ?
RESPONSE: Thank you for this. To provide clarity will identify in text and Figure 2 that this is peak response we are referring to.
Many P2X and P2Y were expressed by the cells, but the use of specific inhibitors shows only a role for P2X4 and P2Y11… can you discuss why the others have no role ?
RESPONSE: The following discussion point appears in the original manuscript “A functional role of P2X1 is not supported here, and corroborates studies in human aveolar macrophage [22].”
Ivermectin is not so easy to use. It is ok if you do patch-clamping on P2X4R-expressing cells, but trickier with other technics. Thus, Ivermectin is a well known inhibitor of SERCA(Ahern 1999), meaning an increase of Ca2+ concentration could be due to a potentiation of P2X4R, but also to a decreased Ca2+ pumping activity of the reticulum.
RESPONSE: Thank you this is a valid point. To this end we had undertaken experiments to show that both PSB-12062 and 5-BDBD inhibited the ivermectin-potentiated response. These results were in the text-based results “Both PSB-12062 and 5-BDBD inhibited the ivermectin potentiated response by 11.5 ±4.5% (N=6) and 16.3 ±3.5% (N=4), respectively.”
What about the fact that some inhibitors increased the Ca2+ mobilization ? That is the case for P2Y2 and P2X1.
RESPONSE: We do not understand the molecular basis for this phenomenon.
Round 2
Reviewer 1 Report
The authors have addressed some of the points I raised, as they modified both the introduction and the methods' sections accordingly.
However, some aspects have not been addressed in the revised manuscript.
In particular:
The authors should discuss how their study differs from other studies in the literature investigating ATP-dependent calcium responses in human macrophages. The authors should indicate in the text that the reason why P2Y4 has not been studied in this paper is the lack of reagents.Author Response
Text has now been included explaining why P2Y4 was not investigated by immunocytochemistry.
We have written a summary at the end of the discussion stating the novelty of this study.
