Type I interferon responses contribute to immune protection against mycobacterial infection
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eLife Assessment
This useful study shows a protective role of type 1 IFN during Mycobacterium tuberculosis infection. It shows that the type 1 IFN response in human skin TST inversely correlates with TB severity, suggesting its protective role. Considering that type I IFN is usually shown to be pro-pathogenic, the higher vulnerability of zebrafish larvae lacking stat2 to M marinum infection is a strong result. However, the conclusion that IFN-I is protective during mycobacterial infection remains indirect and incomplete; the study requires additional mechanistic insights and validation.
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Abstract
Abstract
Type I interferon responses have been considered detrimental to host protection in tuberculosis (TB). We provide novel data to challenge this paradigm, derived from transcriptional profiling of human in vivo immune responses to discover associations with radiographic disease severity in pulmonary TB, combined with mechanistic studies to test causality for observed associations using a zebrafish larval mycobacterial infection model. Type I interferon activity in tissue samples from the site of a standardised mycobacterial challenge, the tuberculin skin test, was associated with less severe human TB disease. Abrogation of type I interferon signalling, by CRISPR-mediated mutagenesis of stat2, led to increased burden and dissemination of Mycobacterium marinum infection in zebrafish larvae. The mechanism for increased severity of mycobacterial infection in zebrafish involves reduced recruitment of myeloid cells required to restrict bacterial growth. Our data support a clear host protective role for type I interferon responses in mycobacterial infection, with potential applications for risk-stratification of adverse outcomes and development of a host-directed therapy to mitigate against severe disease.
Article activity feed
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eLife Assessment
This useful study shows a protective role of type 1 IFN during Mycobacterium tuberculosis infection. It shows that the type 1 IFN response in human skin TST inversely correlates with TB severity, suggesting its protective role. Considering that type I IFN is usually shown to be pro-pathogenic, the higher vulnerability of zebrafish larvae lacking stat2 to M marinum infection is a strong result. However, the conclusion that IFN-I is protective during mycobacterial infection remains indirect and incomplete; the study requires additional mechanistic insights and validation.
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Reviewer #1 (Public review):
Summary:
The manuscript finds a negative relationship between tuberculin skin test-induced type I interferon activity with chest X-ray tuberculosis severity in humans. This evidence is between incomplete and solid. It needs a bioinfomatics/transcriptomics reviewer to make a more insightful judgement. The manuscript demonstrates a convincing role for Stat2 in controlling Mycobacterium marinum infection in zebrafish embryos, incomplete data are presented linking reduced leukocyte recruitment to the infection susceptibility phenotype.
Strengths:
(1) An interesting analysis of TST response correlated with chest X-ray pathology.
(2) Novel data on a protective role for Stat2 in a natural host-mycobacterial species infection pairing.
Weaknesses:
(1) The transcriptional modules are very large sets of genes that do …
Reviewer #1 (Public review):
Summary:
The manuscript finds a negative relationship between tuberculin skin test-induced type I interferon activity with chest X-ray tuberculosis severity in humans. This evidence is between incomplete and solid. It needs a bioinfomatics/transcriptomics reviewer to make a more insightful judgement. The manuscript demonstrates a convincing role for Stat2 in controlling Mycobacterium marinum infection in zebrafish embryos, incomplete data are presented linking reduced leukocyte recruitment to the infection susceptibility phenotype.
Strengths:
(1) An interesting analysis of TST response correlated with chest X-ray pathology.
(2) Novel data on a protective role for Stat2 in a natural host-mycobacterial species infection pairing.
Weaknesses:
(1) The transcriptional modules are very large sets of genes that do not present a clear picture of what is actually being measured relative to other biological pathways.
(2) The link between infection-Stat2-leukocyte recruitment and containment of infection is plausible, but lacks a specific link to the first part of the manuscript.
Major concerns
(1) Line 158: The two transcriptional modules should be placed in the context of other DEG patterns. The macrophage type I interferon module, in particular, is quite large (361 genes). Can this be made more granular in terms of type I IFN ligands and STAT2-dependent genes?
(2) The ifnphi1 injection into mxa:mCherry stat2 crispants is a nice experiment to demonstrate loss of type I IFN responsiveness. Further data is required to demonstrate if important mycobacterial control pathways (IFNy, TNF, il6?, etc) are intact in stat2 crispants before being able to conclude that these phenotypes are specific to type I IFN.
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Reviewer #2 (Public review):
Summary:
This study shows that type I interferon (IFN-I) signaling helps protect against mycobacterial infection. Using human gene expression data and a zebrafish model, the authors find that reduced IFN-I activity is linked to more severe disease. They also show that zebrafish lacking the IFN-I signaling gene stat2 are more vulnerable to infection due to poor macrophage migration. These results suggest a protective role for IFN-I in mycobacterial disease, challenging previous findings from other animal models.
Strengths:
Strengths of the manuscript include the use of human clinical samples to support relevance to disease, along with a genetically tractable zebrafish model that enables mechanistic insight.
Weaknesses:
(1) The manuscript presents intriguing human data showing an inverse correlation between …
Reviewer #2 (Public review):
Summary:
This study shows that type I interferon (IFN-I) signaling helps protect against mycobacterial infection. Using human gene expression data and a zebrafish model, the authors find that reduced IFN-I activity is linked to more severe disease. They also show that zebrafish lacking the IFN-I signaling gene stat2 are more vulnerable to infection due to poor macrophage migration. These results suggest a protective role for IFN-I in mycobacterial disease, challenging previous findings from other animal models.
Strengths:
Strengths of the manuscript include the use of human clinical samples to support relevance to disease, along with a genetically tractable zebrafish model that enables mechanistic insight.
Weaknesses:
(1) The manuscript presents intriguing human data showing an inverse correlation between IFN-I gene signatures and TB disease, but the findings remain correlative and may be cohort-specific. Given that the skin is not a primary site of TB and is relatively immunotolerant, the biological relevance of downregulated IFN-I-related genes in this tissue to systemic or pulmonary TB is unclear.
(2) The reliance on stat2 CRISPants in zebrafish offers a limited view of IFN-I signaling. Including additional crispant lines targeting other key regulators (e.g., ifnar1, tyk2, irf3, irf7) would strengthen the interpretation and clarify whether the observed effects reflect broader IFN-I pathway disruption.
(3) The conclusion that IFN-I is protective contrasts with established findings from murine and non-human primate models, where IFN-I is often detrimental. While the authors highlight species differences, the lack of functional human data and reliance on M. marinum in zebrafish limit the translational relevance. A more balanced discussion addressing these discrepancies would improve the manuscript.
(4) Quantification of bacterial burden using fluorescence intensity alone may not accurately reflect bacterial viability. Complementary methods, such as qPCR for bacterial DNA, would provide a more robust assessment of antimicrobial activity.
(5) Finally, the authors should clarify whether impaired macrophage recruitment in stat2 crispants results from defects in chemotaxis, differentiation, or survival, and address discrepancies between their human blood findings and prior studies.
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Reviewer #3 (Public review):
Summary:
In this manuscript, the authors presented an interesting study providing an insight into the role of Type-I interferon responses in tuberculosis (TB) pathogenesis by combining transcriptome analysis of PBMCs and TST from tuberculosis patients. The zebrafish model was used to identify the changes in the innate immune cell population of macrophages and neutrophils. The findings suggested that Type-I interferon signatures inversely correlated with disease severity in the TST transcriptome data. The authors validated the observations by CRISPR-mediated disruption of stat2 (a critical transcription factor for type I interferon signaling) in zebrafish larvae, showing increased susceptibility to M. marinum infection. Traditionally, type-I interferon responses have been viewed as detrimental in …
Reviewer #3 (Public review):
Summary:
In this manuscript, the authors presented an interesting study providing an insight into the role of Type-I interferon responses in tuberculosis (TB) pathogenesis by combining transcriptome analysis of PBMCs and TST from tuberculosis patients. The zebrafish model was used to identify the changes in the innate immune cell population of macrophages and neutrophils. The findings suggested that Type-I interferon signatures inversely correlated with disease severity in the TST transcriptome data. The authors validated the observations by CRISPR-mediated disruption of stat2 (a critical transcription factor for type I interferon signaling) in zebrafish larvae, showing increased susceptibility to M. marinum infection. Traditionally, type-I interferon responses have been viewed as detrimental in mycobacterial infections, with studies suggesting enhanced susceptibility in certain mouse models. The study tried to identify and further characterize the understanding of the role of type-I interferons in TB.
Strengths:
Traditionally, type-I interferon responses have been viewed as detrimental in mycobacterial infections, with studies suggesting enhanced susceptibility in certain mouse models. The study tried to further understand the role of type-I interferons in TB pathogenesis.
Weaknesses:
Though the study showed an inverse correlation of Type-I interferon with radiological features of TB, the molecular mechanism is largely unexplored in the study, which is making it difficult to understand the basis of the results shown in the manuscript by the authors.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
The manuscript finds a negative relationship between tuberculin skin test-induced type I interferon activity with chest X-ray tuberculosis severity in humans. This evidence is between incomplete and solid. It needs a bioinfomatics/transcriptomics reviewer to make a more insightful judgement. The manuscript demonstrates a convincing role for Stat2 in controlling Mycobacterium marinum infection in zebrafish embryos, incomplete data are presented linking reduced leukocyte recruitment to the infection susceptibility phenotype.
Strengths:
(1) An interesting analysis of TST response correlated with chest X-ray pathology.
(2) Novel data on a protective role for Stat2 in a natural host-mycobacterial species infection pairing.
We appreciate the reviewer’s positive comments.
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
The manuscript finds a negative relationship between tuberculin skin test-induced type I interferon activity with chest X-ray tuberculosis severity in humans. This evidence is between incomplete and solid. It needs a bioinfomatics/transcriptomics reviewer to make a more insightful judgement. The manuscript demonstrates a convincing role for Stat2 in controlling Mycobacterium marinum infection in zebrafish embryos, incomplete data are presented linking reduced leukocyte recruitment to the infection susceptibility phenotype.
Strengths:
(1) An interesting analysis of TST response correlated with chest X-ray pathology.
(2) Novel data on a protective role for Stat2 in a natural host-mycobacterial species infection pairing.
We appreciate the reviewer’s positive comments.
Weaknesses:
(1) The transcriptional modules are very large sets of genes that do not present a clear picture of what is actually being measured relative to other biological pathways.
The transcriptional module analysis is a major strength of our approach. These gene signatures are derived from independent experiments, most of which have been previously published/validated [1,2]. To clarify, they represent co-regulated gene sets downstream of signalling pathways. Increased number of genes in these modules increases their combinatorial specificity for a given biological pathway. In the human data, they serve as orthogonal validation for the bioinformatic analysis showing enrichment of the type I IFN pathway among TST transcriptome genes that are negatively correlated with radiographic disease severity in pulmonary TB (see Figure 2). Importantly, our modules confirm the relationship with type I IFN signalling (see Figure 2E) by discriminating from type II IFN signalling, which is not statistically significantly correlated with radiographic TB severity (see Figure S6C-E).
(2) The link between infection-Stat2-leukocyte recruitment and containment of infection is plausible, but lacks a specific link to the first part of the manuscript.
For clarification, the first part of the study seeks to identify immune response pathways that relate to severity of human disease, leading to the identification of type I IFN signalling. Since the human data are limited to an observational analysis in which we cannot test causality, the second part of our study uses a genetically tractable experimental model to test the hypothesis that type I IFN signalling is host-protective and explore possible mechanisms for a beneficial effect. This leads to the observation that type I IFN responses contribute to early myeloid cell recruitment to the site of infection, that has previously been shown to be crucial for containment of mycobacterial infection in zebrafish larvae. We will further evaluate the introduction and results sections to ensure a clear link between the human and zebrafish work.
Major concerns
(1) Line 158: The two transcriptional modules should be placed in the context of other DEG patterns. The macrophage type I interferon module, in particular, is quite large (361 genes). Can this be made more granular in terms of type I IFN ligands and STAT2-dependent genes?
We respectfully disagree with this comment. For clarification, the 360 gene module reflects the zebrafish larval response to IFNphi1 protein [3]. Type I IFNs are known to induce hundreds of interferon stimulated genes [4]. As explained above, the size of the modules increases specificity for a given signalling pathway. In this case, we are most interested in discriminating type I and type II IFN signalling pathways that represent very different upstream biological processes. The discrimination we achieve with our modular approach is a major advance over previous reports of gene signatures in TB that do not discriminate between the two pathways. In this study, we did not discriminate between signalling downstream of type I IFN ligands and STAT2, consistent with existing literature showing that type I IFN signalling is STAT2 dependent [5,6].
(2) The ifnphi1 injection into mxa:mCherry stat2 crispants is a nice experiment to demonstrate loss of type I IFN responsiveness. Further data is required to demonstrate if important mycobacterial control pathways (IFNy, TNF, il6?, etc) are intact in stat2 crispants before being able to conclude that these phenotypes are specific to type I IFN.
Thank you for the positive comment. We acknowledge this point and will attempt to evaluate whether pro-inflammatory cytokine responses are intact in stat2 CRISPants by qPCR or bulk RNAseq. However, these experiments may prove inconclusive because of the limited sensitivity in this approach.
Reviewer #2 (Public review):
Summary:
This study shows that type I interferon (IFN-I) signaling helps protect against mycobacterial infection. Using human gene expression data and a zebrafish model, the authors find that reduced IFN-I activity is linked to more severe disease. They also show that zebrafish lacking the IFN-I signaling gene stat2 are more vulnerable to infection due to poor macrophage migration. These results suggest a protective role for IFN-I in mycobacterial disease, challenging previous findings from other animal models.
Strengths:
Strengths of the manuscript include the use of human clinical samples to support relevance to disease, along with a genetically tractable zebrafish model that enables mechanistic insight.
We welcome the reviewer’s positive summary of our study.
Weaknesses:
(1) The manuscript presents intriguing human data showing an inverse correlation between IFN-I gene signatures and TB disease, but the findings remain correlative and may be cohort-specific. Given that the skin is not a primary site of TB and is relatively immunotolerant, the biological relevance of downregulated IFN-I-related genes in this tissue to systemic or pulmonary TB is unclear.
We agree with the reviewer that the observational human data are correlative. That is precisely why we extend the study to undertake mechanistic studies in a genetically tractable animal model, using M. marinum infection of zebrafish larvae. In the introduction, we already provide a detailed rationale for the strengths of the TST model to study human immune responses to a standardised mycobacterial challenge. This approach mitigates against the confounding of heterogeneity in bacterial burden and sampling different stages of the natural history of infection in conventional observational human studies. Therefore, the application of the TST is a major strength of this study. We do not understand the context in which the reviewer suggests the skin is immunotolerant. In the present study and previous work we provide molecular level analysis of the TST as a robust cell mediated immune response that reflects molecular perturbation in granuloma from the site of pulmonary TB disease 1.
(2) The reliance on stat2 CRISPants in zebrafish offers a limited view of IFN-I signaling. Including additional crispant lines targeting other key regulators (e.g., ifnar1, tyk2, irf3, irf7) would strengthen the interpretation and clarify whether the observed effects reflect broader IFN-I pathway disruption.
We respectfully disagree with this comment. Our objective was to test the role of type I IFN signalling in M. marinum infection of zebrafish. We show that stat2 deletion effectively disrupts type I IFN signalling (Figure S8). Therefore, we do not see a compelling rationale to evaluate other molecules in the signalling pathway.
(3) The conclusion that IFN-I is protective contrasts with established findings from murine and non-human primate models, where IFN-I is often detrimental. While the authors highlight species differences, the lack of functional human data and reliance on M. marinum in zebrafish limit the translational relevance. A more balanced discussion addressing these discrepancies would improve the manuscript.
We acknowledge that our findings contrast with the prevailing view in published literature to date. We will further review the discussion to see how we can elaborate on the potential strengths and weaknesses of different experimental approaches, which may underpin these discrepancies.
(4) Quantification of bacterial burden using fluorescence intensity alone may not accurately reflect bacterial viability. Complementary methods, such as qPCR for bacterial DNA, would provide a more robust assessment of antimicrobial activity.
We and others have previously validated the use of the quantitative measures of fluorescence, used here as a measure of bacterial load [7,8]. Importantly, our measurements do not rely purely on the total fluorescence signal, but also measures of dissemination of infection, for which we see consistent findings. It is also widely recognised that DNA measurements do not necessarily correlate well with bacterial viability. Therefore, we respectfully disagree that a PCR-based approach will add substantial value to our existing analysis.
(5) Finally, the authors should clarify whether impaired macrophage recruitment in stat2 crispants results from defects in chemotaxis, differentiation, or survival, and address discrepancies between their human blood findings and prior studies.
We acknowledge that these are important questions. Our data show that stat2 disruption does not impact total macrophage numbers at baseline (Figure 4A,B) and therefore do not support any effect of Stat2 signalling on steady state macrophage survival or differentiation. The downregulation of macrophage mpeg1 expression in M. marinum infection precludes long-term follow-up of these cells in the context of infection [9]. Therefore, we cannot currently test the hypothesis that Stat2 signalling may influence death of macrophages recruited to the site of infection or make them more susceptible to the cytopathic effects of direct mycobacterial infection. We will attempt to confirm using short-term time-lapse imaging that cellular migration to the site of hindbrain M. marinum infection is reduced in stat2 deficient zebrafish. On the strength of what is possible to test and the established role of type I IFNs in induction of several chemokines [10,11], the most likely effect is that Stat2 signalling increases recruitment through chemokine production. We are exploring the possibility of testing changes to the chemokine profile in stat2 CRISPants by qPCR or bulk RNAseq, but these experiments may prove inconclusive because of the limitations of sensitivity in this approach.
We recognize that our finding of no relationship between peripheral blood type I IFN activity and severity of human TB contrasts with that of previous studies. As stated in the discussion, the most likely explanation for this is our use of transcriptional modules which reflect exclusive type I IFN responses. The signatures used in other studies include both type I and type II IFN inducible genes and therefore also reflect IFN gamma driven responses.
Reviewer #3 (Public review):
Summary:
In this manuscript, the authors presented an interesting study providing an insight into the role of Type-I interferon responses in tuberculosis (TB) pathogenesis by combining transcriptome analysis of PBMCs and TST from tuberculosis patients. The zebrafish model was used to identify the changes in the innate immune cell population of macrophages and neutrophils. The findings suggested that Type-I interferon signatures inversely correlated with disease severity in the TST transcriptome data. The authors validated the observations by CRISPR-mediated disruption of stat2 (a critical transcription factor for type I interferon signaling) in zebrafish larvae, showing increased susceptibility to M. marinum infection. Traditionally, type-I interferon responses have been viewed as detrimental in mycobacterial infections, with studies suggesting enhanced susceptibility in certain mouse models. The study tried to identify and further characterize the understanding of the role of type-I interferons in TB.
Strengths:
Traditionally, type-I interferon responses have been viewed as detrimental in mycobacterial infections, with studies suggesting enhanced susceptibility in certain mouse models. The study tried to further understand the role of type-I interferons in TB pathogenesis.
We thank the reviewer for their summary.
Weaknesses:
Though the study showed an inverse correlation of Type-I interferon with radiological features of TB, the molecular mechanism is largely unexplored in the study, which is making it difficult to understand the basis of the results shown in the manuscript by the authors.
We respectfully disagree with this comment. The observations in the human data lead to the hypothesis that type I IFN responses may be host-protective, which we then test specifically in the zebrafish model, and explore candidate mechanisms, focussing on myeloid cell recruitment to the site of infection.
References
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(2) Pollara, G., Turner, C.T., Rosenheim, J., Chandran, A., Bell, L.C.K., Khan, A., Patel, A., Peralta, L.F., Folino, A., Akarca, A., et al. (2021). Exaggerated IL-17A activity in human in vivo recall responses discriminates active tuberculosis from latent infection and cured disease. Sci. Transl. Med. 13, eabg7673. https://doi.org/10.1126/scitranslmed.abg7673.
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(5) Blaszczyk, K., Nowicka, H., Kostyrko, K., Antonczyk, A., Wesoly, J., and Bluyssen, H.A.R. (2016). The unique role of STAT2 in constitutive and IFN-induced transcription and antiviral responses. Cytokine Growth Factor Rev. 29, 71–81. https://doi.org/10.1016/j.cytogfr.2016.02.010.
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(7) Stirling, D.R., Suleyman, O., Gil, E., Elks, P.M., Torraca, V., Noursadeghi, M., and Tomlinson, G.S. (2020). Analysis tools to quantify dissemination of pathology in zebrafish larvae. Sci. Rep. 10, 3149. https://doi.org/10.1038/s41598-020-59932-1.
(8) Takaki, K., Davis, J.M., Winglee, K., and Ramakrishnan, L. (2013). Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish. Nat. Protoc. 8, 1114–1124. https://doi.org/10.1038/nprot.2013.068.
(9) Benard, E.L., Racz, P.I., Rougeot, J., Nezhinsky, A.E., Verbeek, F.J., Spaink, H.P., and Meijer, A.H. (2015). Macrophage-expressed perforins mpeg1 and mpeg1.2 have an anti-bacterial function in zebrafish. J. Innate Immun. 7, 136–152. https://doi.org/10.1159/000366103.
(10) Lehmann, M.H., Torres-Domínguez, L.E., Price, P.J.R., Brandmüller, C., Kirschning, C.J., and Sutter, G. (2016). CCL2 expression is mediated by type I IFN receptor and recruits NK and T cells to the lung during MVA infection. J. Leukoc. Biol. 99, 1057–1064. https://doi.org/10.1189/jlb.4MA0815-376RR.
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