Tribbles1 and Cop1 cooperate to protect the host during in vivo mycobacterial infection
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Abstract
Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles 1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo , we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of pro-inflammatory factors il-1 β and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.
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Reply to the reviewers
Manuscript number: RC-2023-02172
Corresponding author(s): Philip Elks
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Reply to the reviewers
Manuscript number: RC-2023-02172
Corresponding author(s): Philip Elks
[The “revision plan” should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.
The document is important for the editors of affiliate journals when they make a first decision on the transferred manuscript. It will also be useful to readers of the reprint and help them to obtain a balanced view of the paper.
If you wish to submit a full revision, please use our "Full Revision" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]
1. General Statements [optional]
This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.
In this paper we report the discovery that a member of the tribbles pseudokinase family, TRIB1 is expressed in human monocytes and is upregulated after stimulation with mycobacterial antigen in a human patient challenge model, the first direct link between immune cell Tribbles expression and innate immune response to infection. We then interrogated the mechanisms of Tribbles roles in TB using a human disease relevant whole-organism in vivo zebrafish model of TB. We show that specifically TRIB1 modulation can tip the battle between host and pathogen enhancing the innate immune response and reducing bacterial burden. We then uncover the molecular mechanisms responsible for the host protective effect of TRIB1, with enhanced antimicrobial reactive nitrogen species and il-1beta, via cooperation with Cop1 E3 ubiquitin ligase. Our findings demonstrate, for the first time, TRIB1 as a host moderator of antimicrobial mechanisms, whose manipulation is of benefit to the host during mycobacterial infection and as such, a potential novel therapeutic target against TB infection.
We thank the reviewers for their positive appraisal of our work and for their helpful suggestions that will improve our manuscript. In particular we would like to highlight the reviewer’s comments on the gap/need for a new zebrafish in vivo model to understand the roles of tribbles in infection that can “be extrapolated into the human system”, and how they feel these findings will be of broad interest and “significance to cross section of the research community” attracting “interest from readers in the fields of infection, immunity, hematology and animal models” alongside “researchers studying all aspects of Tribbles pseudokinase function, especially researchers seeking models to test small molecule agonists and antagonists.”
2. Description of the planned revisions
Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.
Reviewer 1
The major weakness of the manuscript is that the authors do not evaluate C/EBP transcription factors at all. It is rather surprising as they emphasize cooperation between Trib1 and Cop1 in the main title. C/EBP family proteins are key factors of Trib1-mediated modulation of granulocytes and monocytes. Also, slbo, a drosophila homolog of C/EBP, is a target of tribbles, indicating that the pathway is evolutionary conserved. I would request the following experiments and discussions.
RESPONSE: We agree that possible C/EBP roles should be discussed in detail, and we will add a new discussion section on this.
We stand by our data that the host protective mechanism of Trib1 acts requires Cop1, but we are not able to directly show a C/EBP mechanism within the scope of the current project due to a lack of tools/knowledge in the zebrafish on this (further points/comments below on this). It is important to note that we have not claimed a C/EBP mechanism in our manuscript, and we think it is possibly unlikely given that monocyte and granulocyte numbers are not altered after TRIB1 manipulation. Indeed, there are many other candidates other than C/EBP that COP1 could be acting through. Some examples include MAPK (Niespolo et al., Front Immunol, 2020), serine threonine kinases (Durzynska et al., Structure, 2017) and beta-Catenin (Zahid et al., Proteins, 2022).
In response to this comment, we have modified the title from “Tribbles1 and Cop1 cooperate to protect the host during in vivo mycobacterial infection” to “Tribbles1 is host protective during in vivo mycobacterial infection”. We believe our data does show that the protective effect of Tribbles requires Cop1, but changing the title in this way removes any suggestion that they directly cooperate in the potential C/EBP dependent manner, suggested by the reviewer.
Although the authors found the number of neutrophils and monocytes unchanged by Trib1 overexpression nor knockdown, they did not demonstrate the differentiation status of both cell types. This is quite an important issue, given that Trib1 knockout promotes granulocytic differentiation via C/EBPa accumulation in mice. Also, the analysis of granulocytic/monocytic differentiation will provide the crucial information how Trib1 protects the host from mycobacterial infection regulating hematopoietic cell functions. The authors should perform morphological analysis and examine cell surface marker expression to examine whether Trib1 and Cop1 modulates granulocytic and monocytic differentiation with and without Mm infection.
RESPONSE: Unfortunately, we do not have the same level of immunology knowledge nor the antibodies to look at cell surface markers in zebrafish larvae (it is noted that the reviewer identifies that they “not have sufficient expertise in zebrafish models.” We agree with the reviewer that this would be an obvious and informative experiment to do in mouse models, but is not currently possible in zebrafish larval models). The transgenic promoters used (mpx for neutrophils and mpeg1) are robust and widely published to look at total neutrophil and macrophage numbers (Renshaw et al., Blood 2006; Ellett et al., Blood 2011). Mpx, encoding myeloperoxidase, is expressed late in neutrophil differentiation. It is also worth noting that the zebrafish larval model is still a developing organism, and neutrophil/macrophage numbers rise every day between 1 and 5 days post fertilisation, therefore any effect/delay in leukocyte differentiation would likely be captured at the 2dpf timepoint we have already quantified. We cannot perform leukocyte counts during Mm infection reliably as neutrophils/macrophages cluster around infected areas making counting challenging.
However, in response to this comment we will:
- Use a new Tribbles 1 stable CRISPR-Cas9 knockout mutant we have generated and assess neutrophil differentiation using Sudan Black (SD). SD stains neutrophil granules the development of which is during a late phase of neutrophil differentiation.
- Interestingly, it has been shown that a zebrafish myeloid specific C/EBP (c/ebp1) is not required for initial macrophage or granulocyte development, but knockdown does result in a loss of the secondary granule gene LysC (Su et al., Zebrafish, 2007). Therefore, our findings are not inconsistent with existing literature, even if C/EBPs are regulated by Tribbles. However, to test this further we will use an *LysC:mCherry *transgenic line (Buchan et al., PLoS One 2019) to assess expression in developing neutrophils after trib1 manipulation.
It is interesting that Cop1 knockdown zebrafish is viable, given its ubiquitous expression and multiple important targets of protein degradation. The authors should provide the details of phenotype of Cop1 KO larva and discuss on this issue.
RESPONSE: Zebrafish mutants are much less often embryonic lethal than mice as maternally contributed protein stores allow for basic metabolic functions to occur throughout the short period of embryonic development (Rossant and Hopkins. Genes and Development 1992). However, in the case of Cop1 Crispant, this is a knockdown rather than a knockout, so there may be sufficient remaining Cop1 availability for development if it is indeed a requirement for larval viability. Although Cop1 knockout mice are non-viable, hypomorphs are viable and develop relatively normally (similar to our knockdown zebrafish) but are tumour prone as Cop1 is required for effective tumour suppression (Milgliorini et al., JCI, 2011).
We had not commented on the Cop1 larvae phenotype as they look like they develop normally eg. normal body axis, development. However, we agree that this is a relevant point to incorporate into the manuscript and thus will add a comment on this in the Results section. Furthermore, we will add wholebody neutrophil counts into supplementary information, which we have performed and there is no change with cop1 knockdown, suggesting no difference in granulopoiesis.
[Optional] To obtain the more solid evidence for the Cop1 dependent function of Trib1 on mycobacteria infection, it is better to use the Trib1 mutant that loses the Cop1 binding activity. This experiment will strength the authors' conclusion of the Trib1 and Cop1 cooperation.
RESPONSE: We will address this comment by using a newly generated stable zebrafish CRISPR-Cas9 Tribbles 1 knockout line with a 14 base pair deletion that is predicted to lead a premature stop at 94aa in the middle of the pseudokinase domain, lacking the catalytic loop. This also lacks the predicted COP1 binding area at the C terminal of the protein. We will assess bacterial burden in this model.
Previous studies have shown multiple defects in hematopoietic lineages such as M2-like macrophages and eosinophils in Trib1 KO mice, suggesting that Trib1 affects cellular functions of macrophages upon mycobacteria infection. I would request the authors to mention some ideas on this point in discussion.
RESPONSE: We will add a section in the discussion to address this.
Reviewer 2
Structural comparisons are relatively descriptive of identity etc. Nowadays it should be relatively straightforward to comment on structural conservation based on Alphafold models. Specific details may not be accurate but gross folds will be, and comparing those may be more informative.
RESPONSE: We have taken an initial look at Alphafold models and there are indeed structural similarities between zebrafish and human Tribbles. We will incorporate Alphafold structural models and comment on similarities/differences.
Some discussion of the mechanisms regulating TRIB1/2/3 transcriptionally is probably relevant given the differential upregulation observed during infection. There is quite a bit of characterisation of different Tribble promoter regions in humans-how Edoes this translate to Zebrafish?
RESPONSE: We will add a discussion point on what is known about Tribbles promoter regions in humans. We will assess whether anything is known about the promoter regions in zebrafish Tribbles (we have not identified literature on this currently). If nothing is known on this in zebrafish we will attempt to search for regulatory regions found in humans in the zebrafish promoters.
In terms of Crispr use-can it be confirmed that Crispr modified cell lines have effects at the protein level? This is not my specific expertise, but the supplementary evidence shown seems to show some genomic editing is occurring, but not necessarily how it effects protein levels.
RESPONSE: We do not have antibodies that work on zebrafish Tribbles proteins to assess this directly. However, we will address this comment by using a newly generated stable zebrafish CRISPR Tribbles 1 knockout line with a 14 base pair deletion that is predicted to lead a premature stop at 94aa in the middle of the pseudokinase domain, lacking the catalytic loop. Unlike the “CRISPant” knockdown work in the peer-reviewed version, this represents a full knockout of Tribbles 1. We will assess the trib1 cDNA of the full knockout line to assess the knockout in terms of transcript.
A major conclusion of the paper seems to be that TRIB1 works with COP1 in Zebrafish to mediate response to infection. However the discussion does not particularly tie this with the other discussed mechanisms. E.g. JAK/STS, and EBP-linked responses are discussed separately from COP1, where they could well be linked?
RESPONSE: We agree and this comment fits in with some comments from reviewer 1. We will rework areas of the discussion to address this and bring possible mechanisms together into a new discission section.
Reviewer 3
All comments addressed in new revision (see below).
It is noted that this reviewer has “expertise from genetic studies of model organisms to assess all aspects of the tools and approaches used in the paper.”
3. Description of the revisions that have already been incorporated in the transferred manuscript
Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.
Reviewer 1
Figure 1D, E, F is mislabeled in lines 268-271.
RESPONSE: Apologies for this typo, this has now been changed.
Typo in line 399.
RESPONSE: We have changed “suggesting” to “suggest”.
Figure 6A-B is mislabeled in line 415
RESPONSE: Apologies for this typo. We have changed this from “Figure 5A-B” to “Figure 6A-B”.
Reviewer 2
While the protective effect is stated as an effect size 'close to that of HIF-1a', is there additional rationale suggesting that the two may be linked?
RESPONSE: Yes, there have been a number of studies that link Tribbles and Hif1-alpha. The best characterised link is in different cancer cells where Tribbles 3 has been linked to HIF-1alpha or hypoxia (in breast cancer (Wennemers, Breast Cancer Research 2011), renal cell carcinoma cells (Hong et al., Inj J Biol Sci, 2019) and adenocarcinoma (Xing et al., Cancer Management Research, 2020). In Drosophila Hif-1alpha induces TRIB in fat body tissue (Noguchi et al., Genes Cells 2022). We have now added references to these studies to the relevant section in the results.
Reviewer 3
Minor issues: small problems with clarity and figure panel correlation as detailed below:
Mycobacterium marinum Lines 363-365 Refers to Fig 2C-D should be 3C-D
RESPONSE: Apologies for this typo. This has now been changed to 3C-D.
negative controls DN Hif-1alpha and PR (Figure 4A-B). Similarly, trib1 overexpression increased the levels of anti-nitrotyrosine staining, a proxy for immune cell antimicrobial nitric oxide production (Forlenza et al. 2008), to similar levels of DA Hif-1alpha (Elks et al. 2014; Elks et al) Not seeing this for Trib1
RESPONSE: We are not completely sure what the reviewer is referring to here. We think possible confusion stems from the increase of nitric oxide in trib1 is compared to the phenol red control, so we have now clarified that in the text.
As previously observed, overexpression of trib1 significantly reduced bacterial burden compared to phenol red controls when co-injected with tyrosinase guide (Figure 5A-B).
The Fig 3 A-B is correct, although 6A-B appear to be novel panels showing this result
RESPONSE: Yes, we agree, 6A-B has new results showing similar results to 3A-B, as it is necessary to include siblings from the same clutch in each graph to make direct comparisons. To avoid unnecessary confusion, we have removed the “as previously observed” for figure 6 as we had not previously had the tyrosinase co-injection so these are indeed new data.
444 no comma 446 no comma 457 no comma after "activation"
RESPONSE: We have removed these punctuations.
472-475 confusing - better structure in particular in 474 what does "this" refer to?
RESPONSE: We agree, and have clarified in the following new, clarified sentences:
“Lipid droplets form in macrophages during Mtb infection that are potentially used as source of lipids by *Mtb *to allow for intracellular growth (Daniel et al. 2011). However, more recent findings suggest that lipid droplets are formed during the immune activation process after macrophage Mtb infection (Knight et al. 2018), that can subsequently influence the dynamics response of macrophage host defence (Menon et al. 2019). This macrophage lipid metabolism and handling could potentially be influenced by Tribbles.”
525-526 confusing - better structure perhaps begin with 'Because...'
RESPONSE: We have changed this confusing sentence to:
“Here, we demonstrate il-1b and NO control by Trib1, suggesting that Trib1 controls multiple immune pathways and that therapeutic Trib1 manipulation may be more effective than targeting individual immune pathways alone.”
confusing 538 "this and 539 pave the way for further research into TRIB1 as a target for host-derived therapies" Perhaps "further research into TRIB1 as a target for host-derived therapies could potentially improve infection outcome of mycobacterial infection via pharmacological targeted delivery methods and transient manipulation through genetic approaches"
RESPONSE: We have changed this sentence as suggested.
4. Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.
Reviewer 1
The authors should investigate the expression of the C/EBPa protein p42 isoform and/or other C/EBP family proteins such as C/EBPb, and confirm that the p42 is degraded by Trib1 overexpression and recovered by Trib1 and Cop1 knockout. It is also important to determine both p42 and p30 isoforms are preserved in zebrafish.
RESPONSE: This is a complex point to unpick in zebrafish and we believe this to be out of the scope of the current project. We do not claim a link to C/EBP. As mentioned in above comments we think that a link to C/EBP may be unlikely given that monocyte and granulocyte numbers are not altered after TRIB1 manipulation. We will add more data to look at different markers of neutrophils (see above comments). There are many other candidates other than C/EBP that COP1 could be acting through. Some examples include MAPK (Niespolo et al., Front Immunol, 2020), serine threonine kinases (Durzynska et al., Structure, 2017) and beta-Catenin (Zahid et al., Proteins, 2022). There is also evidence suggesting that COP1 and C/EBP have distinct binding sites on TRIB1, potentially unlinking their activity in some biological situations (Murphy et al., Structure, 2015).
C/EBPa is found in zebrafish and is involved in myeloid differentiation and haematopoeisis (Yuan et al., Blood 2011). There is not a huge amount in the literature on this, but it has been shown in zebrafish models that the drug Tanshinone IIA reduces C/EBPa (Park et al., In J Mol Sci, 2017) and we know from previous work in our department that Tanshinone IIA does not affect total neutrophil numbers in the zebrafish larvae (Robertson et al., Sci Trans Medicine, 2014). The most involved C/EBP in zebrafish myelopoiesis appears is a zebrafish specific isoform called c/ebp1 that is myeloid expressed (Lyons et al., Blood 2001). This has a highly conserved carboxy-terminal bZIP domain but the amino-terminal domains are unique. Interestingly, reduction of c/ebp1 does not ablate initial macrophage or granulocyte development, but did result in loss of expression of LysC, a secondary granule marker (we are checking expression of this gene after Trib1 modulation using a LysC:mCherry transgenic zebrafish line).
We do not have antibodies or tools to detect p42 and p30 in zebrafish. As Tribbles1 regulation of C/EBPa appears to be post-translational (Bauer et al., J Clin Invest, 2015), this would be incredibly challenging to unpick in the zebrafish model due to lack of tools to do this. Due to this and the reasons above we believe this to be out of the scope of the current project.
[Optional] The effect of enhanced ERK phosphorylation by Trib1 for the protective effect against mycobacterial infection is another interesting point. It would be better if the authors could provide the ERK phosphorylation status upon Trib1 overexpression.
RESPONSE: Unfortunately, we have no method to answer this question to a conclusive level within the scope of this project. There are limited reports of phosphorylated ERK antibodies that work in wholemount zebrafish (eg, Maurer and Sagerström, BMC Developmental Biology, 2018, that use a rabbit antibody), but this is widely expressed in many tissues of the zebrafish and immune cells would be challenging to resolve.
Reviewer 2
We have addressed or propose to address all of reviewer 2’s comments.
Reviewer 3
We have addressed or propose to address all of reviewer 3’s comments.
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Referee #3
Evidence, reproducibility and clarity
Summary: In this manuscript, the authors test the role of Tribbles psuedokinase 1 in the primary immune defence against Mycobacterium tuberculosis, the pathogen in tuberculosis. After showing increased Trib1 and 2 in response to mycobacterial infection cell culture and from biopsies of challenged human tissue, they turn to a zebrafish model of infection of the caudal vein with Mycobacterium marinum, a natural fish pathogen similar in effects on macrophages to M. tuberculosis in humans.
Authors find that overexpression of tribs 1, 2 and 3 had no strong effect on development but reduced the bacterial burder significantly for Trib1, …
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Referee #3
Evidence, reproducibility and clarity
Summary: In this manuscript, the authors test the role of Tribbles psuedokinase 1 in the primary immune defence against Mycobacterium tuberculosis, the pathogen in tuberculosis. After showing increased Trib1 and 2 in response to mycobacterial infection cell culture and from biopsies of challenged human tissue, they turn to a zebrafish model of infection of the caudal vein with Mycobacterium marinum, a natural fish pathogen similar in effects on macrophages to M. tuberculosis in humans.
Authors find that overexpression of tribs 1, 2 and 3 had no strong effect on development but reduced the bacterial burder significantly for Trib1, less so for Trib2 and not at all for Trib3. Converesely, Trib1 Crisper-mediated knockdown increased Mm burde, which 3 had no effect ( and 2 guide RNAs were not effective at reducing Trib2).
Trib1 increased levels of pro-inflammatory interleukin 1 beta, as measure by a reporter gne , which Trib3 had no similar effect, which was on par to the effect of the Hif-1alpha transcription factor, known to regulate il-1beta. The effect of Trib1 was not upon increased Hif-a (hence independent) but was dependent on the co-factor COP1, a known target of the Trib1 C-tail when activated
No major problems seen
Minor issues: small problems with clarity and figure panel correlation as detailed below Mycobacterium marinum Lines 363-365 Refers to Fig 2C-D should be 3C-D
- 386 negative controls DN Hif-1and PR (Figure 4A-B). Similarly, trib1 overexpression increased
- 387 the levels of anti-nitrotyrosine staining, a proxy for immune cell antimicrobial nitric oxide
- 388 production (Forlenza et al. 2008), to similar levels of DA Hif-1(Elks et al. 2014; Elks et al. Not seeing this for Trib1
- 414 As previously observed, overexpression of trib1 significantly reduced bacterial burden
- 415 compared to phenol red controls when co-injected with tyrosinase guide (Figure 5A-B). The Fig 3 A-B is correct, although 6A-B appear to be novel panels showing this result
444 no comma 446 no comma 457 no comma after "activation" 472-475 confusing - better structure in particular in 474 what does "this" refer to? 525-526 confusing - better structure perhaps begin with 'Because...'
confusing 538 "this and 539 pave the way for further research into TRIB1 as a target for host-derived therapies"
Perhaps "further research into TRIB1 as a target for host-derived therapies could potentially improve infection outcome of mycobacterial infection via pharmacological targeted delivery methods and transient manipulation through genetic approaches"
Significance
General assessment: The paper is well written and clear. The importance of developing host derived therapies is stated well in the introduction and the discussion makes clear the significance of the work to developing the zebrafish as a useful alternative to traditional models for studying the pathophysiology of mycobacterial infection.
Advance: for the Tribbles field, which I can comment on, this advances an important and neglected model organism (the zebrafish), introducing this set of three homologs Trib1, 2 and 3, that are well studied in mammals, Drosophila and C.elegans.
Audience: The paper will be of significance to cross section of the research community on the one hand developing novel approaches to treat TB and on the other to researchers studying all aspects of Tribbles pseudokinase function, especially researchers seeking models to test small molecule agonists and antagonists.
I have expertise from genetic studies of model organisms to assess all aspects of the tools and approaches used in the paper.
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Referee #2
Evidence, reproducibility and clarity
The paper by Hammond et al characterises the role of Tribbles proteins in Zebrafish in response to MTb infection. They show interesting upregulation of TRIB1 and 2, relative to TRIB3 which is not upregulated. This may provide an interesting system to further explore the role of Tribbles in response to infection, which is currently underexplored, but could do with some additional detail to stake that claim more strongly.
Major Comments
Some discussion of the mechanisms regulating TRIB1/2/3 transcriptionally is probably relevant given the differential upregulation observed during infection. There is quite a bit of characterisation of …
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Referee #2
Evidence, reproducibility and clarity
The paper by Hammond et al characterises the role of Tribbles proteins in Zebrafish in response to MTb infection. They show interesting upregulation of TRIB1 and 2, relative to TRIB3 which is not upregulated. This may provide an interesting system to further explore the role of Tribbles in response to infection, which is currently underexplored, but could do with some additional detail to stake that claim more strongly.
Major Comments
Some discussion of the mechanisms regulating TRIB1/2/3 transcriptionally is probably relevant given the differential upregulation observed during infection. There is quite a bit of characterisation of different Tribble promoter regions in humans-how does this translate to Zebrafish?
Structural comparisons are relatively descriptive of identity etc. Nowadays it should be relatively straightforward to comment on structural conservation based on Alphafold models. Specific details may not be accurate but gross folds will be, and comparing those may be more informative.
In terms of Crispr use-can it be confirmed that Crispr modified cell lines have effects at the protein level? This is not my specific expertise, but the supplementary evidence shown seems to show some genomic editing is occurring, but not necessairly how it effects protein levels.
While the protective effect is stated as an effect size 'close to that of HIF-1a', is there additional rationale suggesting that the two may be linked?
Minor comment
A major conclusion of the paper seems to be that TRIB1 works with COP1 in Zebrafish to mediate response to infection. However the discussion does not particularly tie this with the other discussed mechanisms. E.g. JAK/STS, and EBP-linked responses are discussed seperately from COP1, where they could well be linked?
Significance
Tribbles are clearly important in immune cell development. The work is an interesting foray into Tribbles in Zebrafish, which is an interesting tool to be developed going forward. It also hints at a broader role of Tribbles in human infection, but this requires more work to play out.
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Referee #1
Evidence, reproducibility and clarity
Summary
The paper by Hammond et al. describes the protective role of Trib1 for mycobacterial infection such as tuberculosis. They first found that TRIB1 expression is upregulated upon mycobacterial antigen injection in human monocytes. They then tried to perform functional studies using a zebrafish model. To achieve the study, they confirmed the preservation of Trib family genes in zebrafish. The transgenic zebrafish expressing exogenous trib1 demonstrated decreased bacterial burden of M. marium, and trib1 knockdown showed the opposite effect. Trib1 expression also induced the production of Il1B and NO in the Hif independent manner. …
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Referee #1
Evidence, reproducibility and clarity
Summary
The paper by Hammond et al. describes the protective role of Trib1 for mycobacterial infection such as tuberculosis. They first found that TRIB1 expression is upregulated upon mycobacterial antigen injection in human monocytes. They then tried to perform functional studies using a zebrafish model. To achieve the study, they confirmed the preservation of Trib family genes in zebrafish. The transgenic zebrafish expressing exogenous trib1 demonstrated decreased bacterial burden of M. marium, and trib1 knockdown showed the opposite effect. Trib1 expression also induced the production of Il1B and NO in the Hif independent manner. Finally, they found that cop1 knockdown reduced trib1-mediated protection against Mm infection.
Major comments
The major weakness of the manuscript is that the authors do not evaluate C/EBP transcription factors at all. It is rather surprising as they emphasize cooperation between Trib1 and Cop1 in the main title. C/EBP family proteins are key factors of Trib1-mediated modulation of granulocytes and monocytes. Also, slbo, a drosophila homolog of C/EBP, is a target of tribbles, indicating that the pathway is evolutionary conserved. I would request the following experiments and discussions.
- The authors should investigate the expression of the C/EBPa protein p42 isoform and/or other C/EBP family proteins such as C/EBPb, and confirm that the p42 is degraded by Trib1 overexpression and recovered by Trib1 and Cop1 knockout. It is also important to determine both p42 and p30 isoforms are preserved in zebrafish.
- Although the authors found the number of neutrophils and monocytes unchanged by Trib1 overexpression nor knockdown, they did not demonstrate the differentiation status of both cell types. This is quite an important issue, given that Trib1 knockout promotes granulocytic differentiation via C/EBPa accumulation in mice. Also, the analysis of granulocytic/monocytic differentiation will provide the crucial information how Trib1 protects the host from mycobacterial infection regulating hematopoietic cell functions. The authors should perform morphological analysis and examine cell surface marker expression to examine whether Trib1 and Cop1 modulates granulocytic and monocytic differentiation with and without Mm infection.
- It is interesting that Cop1 knockdown zebrafish is viable, given its ubiquitous expression and multiple important targets of protein degradation. The authors should provide the details of phenotype of Cop1 KO larva and discuss on this issue.
- [Optional] The effect of enhanced ERK phosphorylation by Trib1 for the protective effect against mycobacterial infection is another interesting point. It would be better if the authors could provide the ERK phosphorylation status upon Trib1 overexpression.
- [Optional] To obtain the more solid evidence for the Cop1 dependent function of Trib1 on mycobacteria infection, it is better to use the Trib1 mutant that loses the Cop1 binding activity. This experiment will strength the authors' conclusion of the Trib1 and Cop1 cooperation.
Minor comments
- Previous studies have shown multiple defects in hematopoietic lineages such as M2-like macrophages and eosinophils in Trib1 KO mice, suggesting that Trib1 affects cellular functions of macrophages upon mycobacteria infection. I would request the authors to mention some ideas on this point in discussion.
- Figure 1D, E, F is mislabeled in lines 268-271.
- Typo in line 399.
- Figure 6A-B is mislabeled in line 415
Referees cross-commenting
I do not have any cross-comments, however, I believe comments of other reviewers are helpful for authors.
Significance
This study is the first report on the Tribbles protective function for mycobacterial infection. The use of the zebrafish model is unique and provides useful information on tribbles family genes are expressed in hematopoietic cells in zebrafish. As granulocytic and monocytic functions are conserved between fish and mammals, the results can be extrapolated into the human system to understand infection and pathogenic mechanisms of mycobacterium. The lack of investigation on C/EBP transcription factors is a major limitation to be improved in this study, since C/EBPa is a key molecule in the Trib1 and Cop1 cooperation in granulocytic differentiation. The manuscript will attract interest from readers in the fields of infection, immunity, hematology and animal models. This reviewer's field of expertise is hematology/oncology/model animals, although I do not have sufficient expertise in zebrafish models.
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