Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

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1. 

   eLife

   Jun 25, 2021

   Author Response:

   Reviewer #1 (Public Review):

   In this manuscript, the authors build off their previous data where they have identified differences in the sst1 locus as responsible for differences in susceptibility of B6 and C3HeB/Fej mice to Mycobacterium tuberculosis infection. The authors have previously shown that this susceptibility is attributed to higher levels of type I IFN signaling and in particular, the ISG IL-1Ra. The sst1 locus contains many genes that could be contributing to the differential susceptibility in C3HeB/Fej mice, and the model in the field was that differences in Sp110 expression was a likely candidate to explain the susceptibility. However, in this manuscript, the authors show that it is not lower expression of Sp110, but instead decreased expression of another gene in the sst1 locus, Sp140, that …

   Author Response:

   Reviewer #1 (Public Review):

   In this manuscript, the authors build off their previous data where they have identified differences in the sst1 locus as responsible for differences in susceptibility of B6 and C3HeB/Fej mice to Mycobacterium tuberculosis infection. The authors have previously shown that this susceptibility is attributed to higher levels of type I IFN signaling and in particular, the ISG IL-1Ra. The sst1 locus contains many genes that could be contributing to the differential susceptibility in C3HeB/Fej mice, and the model in the field was that differences in Sp110 expression was a likely candidate to explain the susceptibility. However, in this manuscript, the authors show that it is not lower expression of Sp110, but instead decreased expression of another gene in the sst1 locus, Sp140, that contributes to the increased susceptibility of mice carrying the sst1S sequence to bacterial infections. This is a very significant and surprising finding, supported by very clear and convincing data from experiments performed with a high level of rigor. Although identification of the gene responsible for differences in susceptibility and outcomes during bacterial infections is an advance for the field, the manuscript stops there in terms of new insight and falls short of providing any additional information beyond what has already been published regarding how this gene or lucus is functioning to regulate immune responses to infection. This limited scope embodies the major concern for this otherwise strong manuscript.

   We thank for the reviewer for recognizing the importance of our discovery that loss of Sp140 (not Sp110) confers susceptibility to M. tuberculosis. Our generation of Sp140 deficient mice allows us to demonstrate, for the first time, that Sp140 is a negative regulator of type I IFNs. By generating crosses between Sp140–/– and Ifnar–/– mice, we further demonstrate that type I IFNs mediate the susceptibility of Sp140–/– mice to M. tuberculosis and Legionella. The reviewer appears to believe that because IFNs were previously shown to mediate the phenotype of Sst1S mice that somehow the function of Sp140 was already known. By contrast, we feel that in fact the function of Sp140 was not at all clear prior to our work, and that our work does indeed provide important mechanistic insight into the function of Sp140 as a regulator of type I IFNs. Sst1S mice contain many genetic differences compared to B6 mice. It is only because of our work that we can now go back and reinterpret the prior work on Sst1S mice, but this would not be possible without the work we have reported in this paper. Of course we would love to be able to describe more about the molecular mechanism by which Sp140 represses interferon transcription. This is indeed something we are working on. However, our preliminary experiments indicate this is not likely to be straightforward and will require considerable effort that is certainly beyond the scope of this current paper. It should be noted, for example, that Sp140 is in the same protein family as the well-known transcriptional regulator Aire. The mechanism by which Aire regulates gene expression has been studied for almost two decades and is still not entirely clear (and was certainly not clear in the initial foundational paper on Aire function published by Anderson et al in Science in 2002). We expect the mechanism of Sp140 to be similarly complex. Importantly, we now know for the first time which protein to study mechanistically, i.e., SP140 instead of SP110.

   Reviewer #2 (Public Review):

   The authors have suggested the importance of SP140 for resistance to Mtb, Legionella infections in mice. They also provide evidence for IFNaR signalling in mediating the increased susceptibility of SP140-/- mice. While they attribute an important function of the transcriptional regulator SP140 to regulation of type I IFN responses by demonstrating the dysregulation of these responses in the SP140-/- mice, more direct evidence for this is needed.

   We appreciate the reviewer’s succinct summary of the main conclusions of our manuscript. While we would agree that there is more to learn about the mechanism of SP140 function, it is not entirely clear to us what the reviewer means when they say that more “direct” evidence is needed for our claim that Sp140 regulates the IFN response during bacterial infection. We feel that the genetic experiments we provide are clear on this point. The reviewer may be thinking that we are proposing a specific mechanism, e.g., that our model is that Sp140 regulates IFN production by binding to the IFN beta gene; although that is an appealing possibility, we agree that is not shown in our manuscript, and indeed, we are careful not to make any such claim. Indeed, we explicitly state that a more indirect mechanism is possible (line 390). What is clear, though, is that loss of Sp140 mediates susceptibility to infection via (direct or indirect) increases in type I IFN. We observe increased type I IFN responses in Sp140–/– mice in vivo, and moreover, we find that a cross of Sp140–/– mice to Ifnar–/– mice reverses susceptibility to infection. These results demonstrate that the dysregulation of type 1 IFN in the absence of Sp140 is not merely correlative, but in fact drives susceptibility to bacterial infection in vivo.

   Reviewer #3 (Public Review):

   In this manuscript Ji et al carefully examine candidate genes driving a previously described susceptibility within the severe susceptibility to tuberculosis (sst1). Surprisingly, mice deficient in the original candidate gene within this locus, SP110, showed no change in susceptibility to infection with M. tuberculosis. In contrast, the authors found that loss of a second gene in this locus, SP140, recapitulated many phenotypes seen in the SST1 mouse, including increased Type I IFN. SP140 susceptibility was reversed by blocking these exacerbated type I IFNs, similar to SST1 mice. RNAseq analysis identify changes in pro-inflammatory cytokines and type I IFNs. The strengths of this paper are the careful and controlled experiments to target and analyze mouse mutants within a notoriously challenging region with homopolymers. Their results are robust, convincing and will be of broad interest to the field of immunology and host-pathogen interactions. Convincingly identifying a single gene within this region that recapitulates many aspects of the SST1 mouse is very important. While a minor weakness is the lack of any mechanistic understanding of how SP140 functions, this is overcome by the impact of the other findings and it is anticipated that this mouse will now be a key resource to dissect the mechanisms of susceptibility in much greater detail.

   We thank the reviewer for their generous evaluation. Mechanistically, we do show that Sp140 affects resistance to bacterial infection via regulation of the interferon response, which we think is an important and technically non-trivial advance that provides insight into the function of Sp140. However, we agree that the mechanism for how Sp140 regulates type I IFN is not shown (nor is it claimed to be shown) and addressing this mechanism is now an important and exciting question for future studies.

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2. 

   eLife

   Jun 25, 2021

   Evaluation Summary:

   Understanding genes that control susceptibility to infection is critically important. In this manuscript Ji et al. identify the gene SP140 as the driving mutation resulting in susceptibility to a range of bacterial pathogens located within the severe susceptibility to tuberculosis (SST1) in mice. While our understanding of how SP140 functions remains unknown, the identification of a single gene within this locus that regulates Type I IFN is impactful and will be of broad interest for immunologists and microbiologists alike.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
3. 

   eLife

   Jun 25, 2021

   Reviewer #1 (Public Review):

   In this manuscript, the authors build off their previous data where they have identified differences in the sst1 locus as responsible for differences in susceptibility of B6 and C3HeB/Fej mice to Mycobacterium tuberculosis infection. The authors have previously shown that this susceptibility is attributed to higher levels of type I IFN signaling and in particular, the ISG IL-1Ra. The sst1 locus contains many genes that could be contributing to the differential susceptibility in C3HeB/Fej mice, and the model in the field was that differences in Sp110 expression was a likely candidate to explain the susceptibility. However, in this manuscript, the authors show that it is not lower expression of Sp110, but instead decreased expression of another gene in the sst1 locus, Sp140, that contributes to the increased …

   Reviewer #1 (Public Review):

   In this manuscript, the authors build off their previous data where they have identified differences in the sst1 locus as responsible for differences in susceptibility of B6 and C3HeB/Fej mice to Mycobacterium tuberculosis infection. The authors have previously shown that this susceptibility is attributed to higher levels of type I IFN signaling and in particular, the ISG IL-1Ra. The sst1 locus contains many genes that could be contributing to the differential susceptibility in C3HeB/Fej mice, and the model in the field was that differences in Sp110 expression was a likely candidate to explain the susceptibility. However, in this manuscript, the authors show that it is not lower expression of Sp110, but instead decreased expression of another gene in the sst1 locus, Sp140, that contributes to the increased susceptibility of mice carrying the sst1S sequence to bacterial infections. This is a very significant and surprising finding, supported by very clear and convincing data from experiments performed with a high level of rigor. Although identification of the gene responsible for differences in susceptibility and outcomes during bacterial infections is an advance for the field, the manuscript stops there in terms of new insight and falls short of providing any additional information beyond what has already been published regarding how this gene or lucus is functioning to regulate immune responses to infection. This limited scope embodies the major concern for this otherwise strong manuscript.

   Read the original source
4. 

   eLife

   Jun 25, 2021

   Reviewer #2 (Public Review):

   The authors have suggested the importance of SP140 for resistance to Mtb, Legionella infections in mice. They also provide evidence for IFNaR signalling in mediating the increased susceptibility of SP140-/- mice. While they attribute an important function of the transcriptional regulator SP140 to regulation of type I IFN responses by demonstrating the dysregulation of these responses in the SP140-/- mice, more direct evidence for this is needed.

   Read the original source
5. 

   eLife

   Jun 25, 2021

   Reviewer #3 (Public Review):

   In this manuscript Ji et al carefully examine candidate genes driving a previously described susceptibility within the severe susceptibility to tuberculosis (sst1). Surprisingly, mice deficient in the original candidate gene within this locus, SP110, showed no change in susceptibility to infection with M. tuberculosis. In contrast, the authors found that loss of a second gene in this locus, SP140, recapitulated many phenotypes seen in the SST1 mouse, including increased Type I IFN. SP140 susceptibility was reversed by blocking these exacerbated type I IFNs, similar to SST1 mice. RNAseq analysis identify changes in pro-inflammatory cytokines and type I IFNs. The strengths of this paper are the careful and controlled experiments to target and analyze mouse mutants within a notoriously challenging region with …

   Reviewer #3 (Public Review):

   In this manuscript Ji et al carefully examine candidate genes driving a previously described susceptibility within the severe susceptibility to tuberculosis (sst1). Surprisingly, mice deficient in the original candidate gene within this locus, SP110, showed no change in susceptibility to infection with M. tuberculosis. In contrast, the authors found that loss of a second gene in this locus, SP140, recapitulated many phenotypes seen in the SST1 mouse, including increased Type I IFN. SP140 susceptibility was reversed by blocking these exacerbated type I IFNs, similar to SST1 mice. RNAseq analysis identify changes in pro-inflammatory cytokines and type I IFNs. The strengths of this paper are the careful and controlled experiments to target and analyze mouse mutants within a notoriously challenging region with homopolymers. Their results are robust, convincing and will be of broad interest to the field of immunology and host-pathogen interactions. Convincingly identifying a single gene within this region that recapitulates many aspects of the SST1 mouse is very important. While a minor weakness is the lack of any mechanistic understanding of how SP140 functions, this is overcome by the impact of the other findings and it is anticipated that this mouse will now be a key resource to dissect the mechanisms of susceptibility in much greater detail.

   Read the original source
6. 

   Version published to 10.1101/2020.01.07.897553 on bioRxiv

   Jan 8, 2020