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  9. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

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

    In this manuscript, using in vivo infection of Zebrafish embryos with Mycobacterium marinum and THP1-derived macrophages infected with Mycobacterium tuberculosis, the authors show that these pathogenic mycobacteria trigger an increase of K+ concentration through the expression of OXSR1. The ESX1 secretion system that is essential for the virulence of M. marinum is required for the expression of OXSR1 and SPAK. OXSR1 and SPAK are involved in the WNK signaling pathway and are cytoplasmic serine/threonine protein kinases that regulate the function of a series of sodium, potassium and chloride co-transporters via phosphorylation. Given that K+ efflux is now accepted as the main inducer of NLRP3 inflammasome, the authors report that this infection-induced OXSR1 expression restrains the protective NLRP3 inflammasome response leading to IL-1b maturation and secretion. Il-1b as a very potent pro-inflammatory triggers TNF-a production and the authors demonstrate that infection-induced OXSR1 expression suppressed host protective TNF-a and cell death early in fection. It appears therefore that virulent mycobacteria induce OXSR1 expression to reduce inflammasome activation by maintaining high intracellular K+.

    The results presented by the authors are convincing and the conclusions raised by the authors are well supported by the data.

    In zebrafish embryos, OXSR1 knockdown nicely reduces mycobacteria burden. Based on their conclusions that infection-induced OXSR1 expression reduces NLRP3 inflammasome activation, NLRP3 inflammasome activation has therefore a protective effect against bacterial infection. My main concern is that surprisingly, nlrp3 or il1b knockdown has no effect on bacterial burden in comparison to control embryos. Lane 256, as an explanation, the authors wrote "This may have been because we were using mosaic F0 CRISPR knockout, which is not a complete removal". The removal using mosaic F0 CRISPR knockout is nevertheless sufficient to observe a decrease in bacterial burden following OXSR1 knockdown. Would it be possible that OXSR1 also regulates immunity independently of NLRP3 inflammasome?

    Yes, we will add text to the discussion to address potential NLRP3-independent mechanisms that connect OXSR1 to immunity against mycobacterial infection.

    The lack of effect of il1b knockdown on *M. marinum *burden has been corroborated by independent laboratories including a publication from the Elks lab in Journal of Immunology: Ogryzko et al 2019. The Ogryzko study found no effect of il1b knockout on *M. marinum *burden.

    **Other comments:**

    OXSR1 WB in extended Data 3 is really poor quality so that it is hard to see the increased expression of OXSR1 following infection.

    The western blot will be repeated for cleaner images.

    Figure 2C. It is not shown but I guess that similar results should be obtain using M. tuberculosis.

    Material leaving our BSL3 facility must be decontaminated which makes this suggested analysis impossible in our facility.

    Figures 5D and 5E. To confirm the involvement of NLRP3, in addition of using MCC950, NLRP3 knock down using siRNA should be also performed. NLRP3-deficient THP-1 cells are also commercially available if the siRNA-mediated knock down of NLRP3 is not convincing enough.

    We will purchase NLRP3 deficient THP-1 cells and use our existing shRNA vector to create NLRP3 and OXSR1 deficient cells. We will repeat the experiments in 5D and 5E in these cells to confirm NLRP3 involvement.

    **Minor comments:**

    How do the authors think that mycobacterium induces OXSR1 expression following infection? It has not been investigated and it is not discussed.

    In Fig1A we showed upregulation of *oxsr1a *transcription and in Fig2A we showed upregulation of OXSR1 protein. In line 204 of the discussion we described our hypothesis that *oxsr1a *transcription is responsive to the mycobacterial ESX1 secretion system.*

    Reviewer #1 (Significance (Required)):

    The observations reported in this manuscript are interesting since for the first time, it is described that virulent mycobacteria induce OXSR1 expression to reduce NLRP3 inflammasome activation by maintaining high intracellular K+. This is quite a significant advance in the field. To escape immune control, many successful intracellular pathogens have evolved methods to limit inflammasome activation. While it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux and inflammasome activation was not explored.

    My field of expertise is the regulation of inflammasome activation. As far as I remember, I've never reviewed a paper using zebrafish embryos but here, the explanations and data are clear so that it was easy to understand and to evaluate. Likewise, I did not know the WNK signaling pathway but the literature clearly shows that it is involved in intracellular ionic balance.

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

    Hortle et al, in this study evaluated the role of WNK kinases SPAK and OXSR1 during M. marinum and M. tuberculosis infection. These two kinases inhibit the KCC channels which have a tendency to export potassium out of the cell. Since potassium efflux is a known stimulator of NLRP3 inflammasome activation, this raises the possible role of these kinases in inflammation and infection. Authors showed that inhibiting OXSR1 genetically and chemically reduced the mycobacterium survival in cells and zebrafish model, thus proposing OXSR1 as a host-directed therapeutic candidate. They showed that knockdown of OXSR1a leads to NLRP3 inflammasome mediated IL1B induction, which results in increase in TNFa and suppression of mycobacterium growth. Furthermore, reduction in mycobacterium growth in OXSR1a KD zebrafish embryos was found to be dependent on ESX1 machinery of Mycobacterium.

    The role of potassium in regulating Mycobacterium host response is novel. However there are few things which are missing from this interesting work.

    **Main comments**

    1. Since OXSR1 is known to inhibit KCC channels, which will lead to increase intracellular potassium. Why in infected control cells there is no increase potassium, Fig 2C. What would be the role of potassium in OXSR1 mediated control of Mtb growth?

    We will perform more experiments with altered levels of extracellular potassium to determine if infected control cells have increased intracellular potassium compared to OXSR1 knockdown cells.

    Does addition of extracellular potassium restricts mycobacterium in OXSR1-KD cells?

    We will perform additional experiments with the addition of potassium to the cell culture medium to address this concern.

    Since OXSR1 is known to inhibit KCC channels, What happens to the activity of these channels in OXSR1 KD cells? This is important, because authors could not find any difference in intracellular potassium between uninfected control and uninfected OXSR1 KD cells (Fig 2C). It will be good to add the flowcytometric histogram or dot plots of potassium staining in the main figure or in extended figures.

    We have data showing that although there is minimal difference in basal K+ level in OXSR1 KD cells, there is significantly lower K+ level when the cells are placed in High K+ media, or osmotic shock. We will include this data in the revised manuscript. We will amend the figures to include Flow plots.

    Acquisition of potassium stained cells - In methodology it has been mentioned that ion K+ Green stained undifferentiated THP1 cells were acquired using PE channel while differentiated THP1 cells were acquired using FITC channel. Furthermore in methods its mentioned that Leica Sp8 microscope was used to acquire images, however I do not see any of this data in the manuscript.

    Ion K+ green emits into both the PE and FITC channels. Our choice to use the FITC or PE channel depended on whether the cells were also infected with red fluorescent bacteria which “contaminates” the PE channel.

    Fig 2E and 3D - Meaning of "Normalized CFU/ml"? Each dot represents what? How many times this experiment was performed, please add in the legend.

    Normalized CFU/ml means that the CFU at 3 day post infection were normalized to the 0 day post infection intracellular bacterial burden, to adjust for any differences in phagocytosis of bacteria. Each dot represents the CFU from an infected well in a single representative experiment and the experiment was repeated 3 times. This information will be added to the figure legend.


    Fig 1D - What could be the reason of no statistical significant difference between wild type and homozygous oxsr1a-KO fish?

    This data is from two experimental replicates. We are currently growing more breeding fish to generate embryos for experimental replicates.

    Good to have a schematic model showing the finding s of the study

    We will add a schematic model to the manuscript.

    TNFa is double edge sword and can lead to pathology. Hence treatment of chronically infected animals (say mice) by Compound B, will be needed to confirm the HDT activity of OXSR1.

    Yes, we will add discussion of this point as a caveat to our future direction of using OXSR1 inhibition as a HDT.

    Reviewer #2 (Significance (Required)):

    This study showed role of kinases, which regulate trafficking of potassium, in mycobacterium-host interaction. Since kinases are draggable, so this opens a new area for developing host-directed therapies for TB.

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

    In this study, the authors suggest to have evidence for OXSR1 to inhibit NLRP3 inflammasome activation by limiting potassium efflux during mycobacterial infection. To my opinion, the study lacks important results supporting their main conclusions. In many instances, the authors have over-interpreted their data and I therefore do not support publication of this study.

    **Main comments:**

    Activation of the NLRP3 inflammasome upon OXSR1 knockdown was not convincingly demonstrated.

    We will address the activation state of the NLRP3 inflammasome with NLRP3 KO and OXSR1 KD cells as also suggested by reviewer 1: We will purchase NLRP3 deficient THP-1 cells and use our existing shRNA vector to create NLRP3 and OXSR1 deficient cells. We will repeat the experiments in 5D and 5E in these cells to confirm NLRP3 involvement.

    Clearance of bacteria in an organism, herein zebrafish, involves mechanisms in different cell types including downstream of inflammasome activation. Thus, bacterial clearance experiments in THP-1 cells might not necessarily be related to in vivo experiments in an organismal context. Finally, a mechanism as to how mycobacteria enhance OXSR1 expression to block a NLRP3-mediated response has not been addressed.

    We are not able to perform in depth analysis of the bacterial side of this host-pathogen interaction as my lab will close in the next 4 months. We have shown that transcriptional upregulation of *oxsr1a *is ESX1-dependent. We will include data on OXSR1 protein expression with WT and ESX1 mutant bacteria when we repeat the western blots in Extended data 3.

    **Specific comments:**

    1. The author showed that the M. marinum ESX1 secretion system induced OXSR1 expression to inhibit the NLRP3 inflammasome activation. This is contradictory to another recent study (PMID: 18852239), which showed that the ESX1 secretion system activated the NLRP3 inflammasome. __These effects are not mutually exclusive. The ESX1 secretion system has a “deliberate” purpose in exporting mycobacterial effector proteins to subvert cellular immunity while also having an “accidental” role in exposing the host cell cytosol to vacluolar contents that can activate cellular immunity. We do not assert that mycobacteria completely inhibit all NLRP3 activation – rather that attempts to stop full activation via inducing the expression of host OXSR1. This can be seen in the IL-1b data in figure 3E, where infected WT cells release more IL-1b than MCC950 treated cells, but less than OXSR1 KD cells. __

    In line 102, based on Data shown in Fig 1D, the authors concluded that homozygous, but not heterozygous, oxsr1asyd5 embryos showed reduced bacterial burden. However, in Fig 1D, the difference among the genotypes is not significant.

    This concern will be addressed with additional replicates.

    In line 196, the authors stated that "We present evidence that pathogenic mycobacteria increase macrophage K+ concentration by inducing expression of OXSR1." However, the authors did not provide evidence for this.

    We will soften this phrase in the discussion to replace “by inducing” with “and induce”.

    Based on Extended data 3, the authors concluded that infection increases the expression of OXSR1. However, this is not evidenced in the Western Blot. In addition, in panel B, the OXSR1 blot showed many non-specific bands with decreased intensity in OXSR1 knockdown conditions suggesting that there is unequal protein loading making it impossible to interpret these results.

    We will repeat the western blots as per Reviewer 1’s comment as well.

    The authors concluded that infection-induced OXSR1 expression suppressed inflammasome activity to aid mycobacterial infection. Experiments with Compound B, that inhibits OXSR1 phosphorylation, are used in support of the above conclusion. I do not really see a connection between OXSR1 expression and the inhibitor experiment.

    We will reword “expression” to “activity” in regards to the inhibitor experiment.

    In line 187, "Knockdown of tnfa reduced the amount of infection-induced tnfa promoter-driven GFP produced around sites of infection ....". How can a knockdown of tnfa affect the GFP expression driven by the tnfa promoter ?

    The promoter fragment used in the TgBAC construct contains target sites for two of our guide RNAs. We will also include qPCR validation of the knockdown.

    Reviewer #3 (Significance (Required)):

    Mechanism underlying decreased intracellular potassium level is of great interest in the inflammasome field. However, their observation is not in line with published studies.

    Audience in the pathogen-host interaction field will be interested.

    Expertise: dissection of signalling pathway regulation, molecular and cellular mechanism underlying NLRP3 inflammasome activation. We are not using zebrafish model.

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  10. 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

    In this study, the authors suggest to have evidence for OXSR1 to inhibit NLRP3 inflammasome activation by limiting potassium efflux during mycobacterial infection. To my opinion, the study lacks important results supporting their main conclusions. In many instances, the authors have over-interpreted their data and I therefore do not support publication of this study.

    Main comments:

    Activation of the NLRP3 inflammasome upon OXSR1 knockdown was not convincingly demonstrated. Clearance of bacteria in an organism, herein zebrafish, involves mechanisms in different cell types including downstream of inflammasome activation. Thus, bacterial clearance experiments in THP-1 cells might not necessarily be related to in vivo experiments in an organismal context. Finally, a mechanism as to how mycobacteria enhance OXSR1 expression to block a NLRP3-mediated response has not been addressed.

    Specific comments:

    1. The author showed that the M. marinum ESX1 secretion system induced OXSR1 expression to inhibit the NLRP3 inflammasome activation. This is contradictory to another recent study (PMID: 18852239), which showed that the ESX1 secretion system activated the NLRP3 inflammasome.
    2. In line 102, based on Data shown in Fig 1D, the authors concluded that homozygous, but not heterozygous, oxsr1asyd5 embryos showed reduced bacterial burden. However, in Fig 1D, the difference among the genotypes is not significant.
    3. In line 196, the authors stated that "We present evidence that pathogenic mycobacteria increase macrophage K+ concentration by inducing expression of OXSR1." However, the authors did not provide evidence for this.
    4. Based on Extended data 3, the authors concluded that infection increases the expression of OXSR1. However, this is not evidenced in the Western Blot. In addition, in panel B, the OXSR1 blot showed many non-specific bands with decreased intensity in OXSR1 knockdown conditions suggesting that there is unequal protein loading making it impossible to interpret these results.
    5. The authors concluded that infection-induced OXSR1 expression suppressed inflammasome activity to aid mycobacterial infection. Experiments with Compound B, that inhibits OXSR1 phosphorylation, are used in support of the above conclusion. I do not really see a connection between OXSR1 expression and the inhibitor experiment.
    6. In line 187, "Knockdown of tnfa reduced the amount of infection-induced tnfa promoter-driven GFP produced around sites of infection ....". How can a knockdown of tnfa affect the GFP expression driven by the tnfa promoter ?

    Significance

    Mechanism underlying decreased intracellular potassium level is of great interest in the inflammasome field. However, their observation is not in line with published studies.

    Audience in the pathogen-host interaction field will be interested.

    Expertise: dissection of signalling pathway regulation, molecular and cellular mechanism underlying NLRP3 inflammasome activation. We are not using zebrafish model.

    Read the original source
    Was this evaluation helpful?
  11. 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

    Hortle et al, in this study evaluated the role of WNK kinases SPAK and OXSR1 during M. marinum and M. tuberculosis infection. These two kinases inhibit the KCC channels which have a tendency to export potassium out of the cell. Since potassium efflux is a known stimulator of NLRP3 inflammasome activation, this raises the possible role of these kinases in inflammation and infection. Authors showed that inhibiting OXSR1 genetically and chemically reduced the mycobacterium survival in cells and zebrafish model, thus proposing OXSR1 as a host-directed therapeutic candidate. They showed that knockdown of OXSR1a leads to NLRP3 inflammasome mediated IL1B induction, which results in increase in TNFa and suppression of mycobacterium growth. Furthermore, reduction in mycobacterium growth in OXSR1a KD zebrafish embryos was found to be dependent on ESX1 machinery of Mycobacterium.

    The role of potassium in regulating Mycobacterium host response is novel. However there are few things which are missing from this interesting work.

    Main comments

    1. Since OXSR1 is known to inhibit KCC channels, which will lead to increase intracellular potassium. Why in infected control cells there is no increase potassium, Fig 2C. What would be the role of potassium in OXSR1 mediated control of Mtb growth? Does addition of extracellular potassium restricts mycobacterium in OXSR1-KD cells?
    2. Since OXSR1 is known to inhibit KCC channels, What happens to the activity of these channels in OXSR1 KD cells? This is important, because authors could not find any difference in intracellular potassium between uninfected control and uninfected OXSR1 KD cells (Fig 2C). It will be good to add the flowcytometric histogram or dot plots of potassium staining in the main figure or in extended figures.
    3. Acquisition of potassium stained cells - In methodology it has been mentioned that ion K+ Green stained undifferentiated THP1 cells were acquired using PE channel while differentiated THP1 cells were acquired using FITC channel. Furthermore in methods its mentioned that Leica Sp8 microscope was used to acquire images, however I do not see any of this data in the manuscript.
    4. Fig 2E and 3D - Meaning of "Normalized CFU/ml"? Each dot represents what? How many times this experiment was performed, please add in the legend.
    5. Fig 1D - What could be the reason of no statistical significant difference between wild type and homozygous oxsr1a-KO fish?
    6. Good to have a schematic model showing the finding s of the study
    7. TNFa is double edge sword and can lead to pathology. Hence treatment of chronically infected animals (say mice) by Compound B, will be needed to confirm the HDT activity of OXSR1.
      

    Significance

    This study showed role of kinases, which regulate trafficking of potassium, in mycobacterium-host interaction. Since kinases are draggable, so this opens a new area for developing host-directed therapies for TB.

    Read the original source
    Was this evaluation helpful?
  12. 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

    In this manuscript, using in vivo infection of Zebrafish embryos with Mycobacterium marinum and THP1-derived macrophages infected with Mycobacterium tuberculosis, the authors show that these pathogenic mycobacteria trigger an increase of K+ concentration through the expression of OXSR1. The ESX1 secretion system that is essential for the virulence of M. marinum is required for the expression of OXSR1 and SPAK. OXSR1 and SPAK are involved in the WNK signaling pathway and are cytoplasmic serine/threonine protein kinases that regulate the function of a series of sodium, potassium and chloride co-transporters via phosphorylation. Given that K+ efflux is now accepted as the main inducer of NLRP3 inflammasome, the authors report that this infection-induced OXSR1 expression restrains the protective NLRP3 inflammasome response leading to IL-1b maturation and secretion. Il-1b as a very potent pro-inflammatory triggers TNF-a production and the authors demonstrate that infection-induced OXSR1 expression suppressed host protective TNF-a and cell death early in fection. It appears therefore that virulent mycobacteria induce OXSR1 expression to reduce inflammasome activation by maintaining high intracellular K+.

    The results presented by the authors are convincing and the conclusions raised by the authors are well supported by the data.

    In zebrafish embryos, OXSR1 knockdown nicely reduces mycobacteria burden. Based on their conclusions that infection-induced OXSR1 expression reduces NLRP3 inflammasome activation, NLRP3 inflammasome activation has therefore a protective effect against bacterial infection. My main concern is that surprisingly, nlrp3 or il1b knockdown has no effect on bacterial burden in comparison to control embryos. Lane 256, as an explanation, the authors wrote "This may have been because we were using mosaic F0 CRISPR knockout, which is not a complete removal". The removal using mosaic F0 CRISPR knockout is nevertheless sufficient to observe a decrease in bacterial burden following OXSR1 knockdown. Would it be possible that OXSR1 also regulates immunity independently of NLRP3 inflammasome?

    Other comments:

    OXSR1 WB in extended Data 3 is really poor quality so that it is hard to see the increased expression of OXSR1 following infection.

    Figure 2C. It is not shown but I guess that similar results should be obtain using M. tuberculosis.

    Figures 5D and 5E. To confirm the involvement of NLRP3, in addition of using MCC950, NLRP3 knock down using siRNA should be also performed. NLRP3-deficient THP-1 cells are also commercially available if the siRNA-mediated knock down of NLRP3 is not convincing enough.

    Minor comments:

    How do the authors think that mycobacterium induces OXSR1 expression following infection? It has not been investigated and it is not discussed.

    Significance

    The observations reported in this manuscript are interesting since for the first time, it is described that virulent mycobacteria induce OXSR1 expression to reduce NLRP3 inflammasome activation by maintaining high intracellular K+. This is quite a significant advance in the field. To escape immune control, many successful intracellular pathogens have evolved methods to limit inflammasome activation. While it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux and inflammasome activation was not explored.

    My field of expertise is the regulation of inflammasome activation. As far as I remember, I've never reviewed a paper using zebrafish embryos but here, the explanations and data are clear so that it was easy to understand and to evaluate. Likewise, I did not know the WNK signaling pathway but the literature clearly shows that it is involved in intracellular ionic balance.

    Read the original source
    Was this evaluation helpful?