Interferon-β induction heterogeneity during KSHV infection is correlated to levels and activation of the transcription factors ATF2 and RelA, and not IRF3

  1. Machika Kaku
  2. Marta Maria Gaglia

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Careful regulation of type I interferons (IFN) is vital for balancing tissue damage and protection against infections. We previously found that during Kaposi’s sarcoma-associated herpesvirus infection, type I IFN induction was limited to a small percentage of infected cells. This heterogeneity was not explained by viral gene expression. Here, we used a fluorescent reporter and fluorescence activated cell sorting to investigate the source of this heterogeneity. Surprisingly, the canonical IFN induction pathway culminating in the activation of the IRF3 transcription factor was similarly activated between cells that made high vs. low/no IFN-β. In contrast, the activation or expression of the two other IFN transcription factors, the NF-κB subunit RelA and the AP-1 subunit ATF2, correlated with IFN-β induction. Our results suggest that during viral infection, activation of IRF3 does not automatically result in IFN responses at the level of individual cells, but that other factors, such as NF-κB and AP-1, are limiting for type I IFN induction.

Importance

The ability of mammalian cells to react to viral infections is a crucial step in the induction of immune responses. The first course of action for the cell is to express and release type I interferons like interferon-β (IFN-β), secreted molecules that warn surrounding cells. Single-cell level examination of gene expression has revealed that surprisingly, during many viral infections, only a small fraction of infected cells makes IFN-β. This is likely a mechanism to prevent immune system overreactions. However, it remains unclear why only some cells respond. Here, we find that during infection with Kaposi’s sarcoma-associated herpesvirus, an oncogenic virus that affects immunocompromised individuals, the transcription factors AP-1 and NF-κB, rather than the more commonly studied IRF3, may decide which cells go on to make IFN-β. Our findings contribute to a better understanding of complex gene regulation and shed light on a process that fights an oncogenic virus.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Response to the reviewers

    We thank the reviewers for recognizing the importance of study, and how it “addresses a long-standing question in the heterogeneity of cellular responses to stressors”, “makes a conceptual advance by identifying transcription factors as the limiting determinant of IFN-β induction in KSHV-infected cells”, and “serves as a crucial starting point for understanding cellular heterogeneity”. We agree that our findings appeal to a broad audience interested in virology, immunology, cell biology, and gene transcription.

    We also thank the reviewers for their insightful suggestions that will greatly strengthen our study. Below we detail how we plan to address their comments experimentally and how we have already edited the text to respond to them.

    Referee #1

    One experiment that may provide some insight into the selective RelA activation is to quantify viral genomes within the high and low IFN producing cells. Perhaps, the genome as a PAMP, is more abundant in the inducing cells.

    We have added a note in the Discussion section (line 417) that we have evidence that the cGAS PAMP in our system is mitochondrial DNA, not viral DNA. Moreover, our results suggest that the variation in PAMP levels are not the source of heterogeneity, as this would cause heterogeneous activation of the cGAS-STING-TBK1-IRF3 axis. Instead, we have discovered that TBK1 and IRF3 are activated even in cells without interferon-β induction.

    Referee #2

    1) While the study presents intriguing evidence for AP-1 involvement in regulating IFN-β responses, the reliance on total c-Jun levels as a readout is limiting. Because c-Jun activity depends on phosphorylation and promoter binding, additional experiments (i.e., phospho-c-Jun analysis or ChIP at the IFNB1 promoter) would strengthen the link between AP-1 activity and the observed reporter outcomes.

    We agree that that a stronger link between AP-1 activity and IFN induction would improve our study, so we have cloned interferon-β reporter constructs that contain mutations in the AP-1 binding sites. We plan to use this reporter, as well as IFN-β reporter constructs that contain mutations in either the AP-1, IRF3, or NF-κB binding sites, to mechanistically test the connection between AP-1 and activation of the IFN promoter. As a control, we will test that the mutations block reporter induction after stimulation with a well characterized agonist of the IFN induction pathway such as poly(I:C). We have previously investigated c-Jun and ATF2 phosphorylation during KSHV reactivation and caspase inhibition. Surprisingly, in preliminary experiments we did not detect phosphorylation of either AP-1 subunit. We will confirm this result and add these data to the manuscript.

    2) The data presented demonstrating that Serine 386 phosphorylation does not distinguish first responder cells is strong. Including complementary data on Ser396 phosphorylation would strengthen the conclusion, as this well-established activation marker is readily detectable with available reagents and would help confirm that the potentiation of IRF3 activity is not the driver of the observed heterogeneity.

    We will complement the Ser386 results with Ser396 staining.

    3) Consider updating the title to more directly reflect the findings (e.g. "Interferon-β induction heterogeneity during KSHV infection is correlated to expression of ATF2 and RelA")

    We have updated the title to “Interferon-β induction heterogeneity during KSHV infection is correlated to levels and activation of the transcription factors ATF2 and RelA, and not IRF3”

    *4) To ease the interpretation of data, indicate what the black and white circles indicate in the figure legends. *

    We have updated the figures to be more intuitive, using + and -.

    5) IE ORF50 is used to show no differences between first responders and non-responders, but showing early and late genes across tdTomato positive and negative cells would rule out potential differences in progression through reactivation.

    We added a clarification in the Results section (line 195), explaining that we have examined the progression of viral reactivation through single-cell transcriptomics in our previous publication, and that the results indicate that viral gene expression plays a small role in interferon-β heterogeneity. We favor the scRNA-seq dataset for this conclusion, because the tdTomato negative cell population represents a mix of non-reactivating cells, which would not be expected to make IFN, and reactivating cells that fail to induce IFN expression.

    6) The data in Figure 5D (quantified in E and F) show a compelling trend. This could be further clarified by plotting a trend line that connects the results of independent experiments, rather than only showing individual data points. Such visualization would make the consistency of the observed trend across experiments more apparent.

    We have added lines in the graphs in Figure 5 to ease visualization.

    Referee #3

    A major worry comes from using lentiviral transduction to insert the reporter promoter into cells without selecting for clones. Lentiviral transduction introduces heterogeneity due to random insertion of their vector. This results in different copy numbers for the reporter construct, leading to heterogeneity in the reporter expression. Additionally, the expression of foreign proteins, particularly in immune cells, can be perceived as danger signals (10.1007/s12015-016-9670-8) and occasionally trigger p65 activation. To control for this, the authors could validate their reporter results by including a non-IFNb promoter (e.g., constitutive) expressing tdTomato and verifying that these cell populations do not also express endogenous IFNb mRNAs.

    We did not select clonal cell lines because different cells may have different reactivation propensity. Moreover, we did not want the tdTomato signal to reflect specific regulation of a single genomic region. We have now added an explanation as to why we did not clonally select that cell lines in the Results section (line 157). Our control conditions that do not result in IFN-β induction show that lentiviral insertion is not sufficient to cause IFN induction, as we did not detect IFN-β mRNA in the untreated reporter cells (first bar in Figure 1C). We also clarified in the Results section (line 184) that the selective enrichment of both IFN-β and tdTomato mRNA in the sorted tdTomato+ cells demonstrates that tdTomato is a faithful reporter for rare IFN-β expression, regardless of heterogeneous lentiviral transduction in the population. To further verify that lentiviral transduction does not play a role in introducing heterogeneity in induction of our tdTomato reporter and of IFN-β, we will measure IFN-β levels in BC-3 cells constitutively expressing tdTomato, which we have already created. We may also sort BC-3 cells constitutively expressing tdTomato and check that the tdTomato signal is not predictive of IFN expression in these cells. However, the expectation is that all or most cells will be tdTomato positive, which may make sorting for tdTomato negative cells impossible.

    Regarding AP1 and NF-kB activation, the authors could investigate downstream genes such as GADD45B, HSPA1A, and ATF-3 (for AP1), and IL-6, TNFAIP3 (A20) (for both AP1 and NFkB). It would be interesting to determine if these genes are exclusively expressed in tdTomato-expressing cells.

    We will quantify the mRNA levels of these genes by performing qPCR on our cDNA from sort experiments. So far, we have detected IL-6 induction but no enrichment of this transcript in the sorted tdTomato+ samples.

    While the authors observed no direct correlation between c-Jun alone and IFN-b production, it is conceivable that TPA-induced c-Jun primes the cells that become fully transcriptionally active upon a stimulus like viral reactivation. I propose that the authors attempt to inhibit c-Jun activation during KSHV reactivation (TPA + caspase inhibitor) using inhibitors like SP600125 and subsequently assess whether this blockade reduces the proportion of IFNb+ cells.

    We have tried using the suggested inhibitor (SP600125), but found that it inhibits KSHV reactivation, making any result on IFN levels difficult to interpret. Currently, we are testing a dual AP-1 and NF-κB inhibitor (SP100030) and may add these data to the results if we do not encounter similar issues.

  2. Review Commons

    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 #3

    Evidence, reproducibility and clarity

    Summary

    Kaku and Gaglia's study provides one step further in the ongoing debate surrounding viral versus innate immune heterogeneity. They addressed this question by creating a reporter B cell line (BC-3) that expresses tdTomato under the IFNb promoter. This particular cell line is known to be latently infected with KSHV, and lytic infection can be induced by treatment with the protein kinase C activator TPA.

    Through FACS sorting upon KSHV lytic infection, the authors observed a correlation between the promoter reporter activation and endogenous IFNb and IFNl mRNA levels. However, this correlation did not extend to viral replication, indicating that only a fraction of virus-infected cells produce IFN. They identified these cells as "first responders" upon viral replication by treating them with anti-IFNAR, confirming that IFN production is triggered by the cGAS-STING pathway sensing lytic virus infection.

    Surprisingly, p-IRF3 activation was not limited to IFN-producing cells, suggesting the involvement of other key transcription factors. Indeed, they found a correlation between NF-kB and AP1 activation and IFN production. The study concludes that the combined action of NF-kB, AP1, and IRF3 is crucial for robust IFN production.

    Major comment

    The author effectively dissects the necessary components for IFNb activation, despite acknowledging the limitations of their findings. All my potential anecdotal queries, such as the role of other viruses or agonists and the treatment of cells with NF-kB inhibitors, are thoroughly addressed in their discussion.

    However, a major worry comes from using lentiviral transduction to insert the reporter promoter into cells without selecting for clones. Lentiviral transduction introduces heterogeneity due to random insertion of their vector. This results in different copy numbers for the reporter construct, leading to heterogeneity in the reporter expression. Additionally, the expression of foreign proteins, particularly in immune cells, can be perceived as danger signals (10.1007/s12015-016-9670-8) and occasionally trigger p65 activation. To control for this, the authors could validate their reporter results by including a non-IFNb promoter (e.g., constitutive) expressing tdTomato and verifying that these cell populations do not also express endogenous IFNb mRNAs.

    Minor comments

    Regarding AP1 and NF-kB activation, the authors could investigate downstream genes such as GADD45B, HSPA1A, and ATF-3 (for AP1), and IL-6, TNFAIP3 (A20) (for both AP1 and NFkB). It would be interesting to determine if these genes are exclusively expressed in tdTomato-expressing cells.

    While the authors observed no direct correlation between c-Jun alone and IFN-b production, it is conceivable that TPA-induced c-Jun primes the cells that become fully transcriptionally active upon a stimulus like viral reactivation. I propose that the authors attempt to inhibit c-Jun activation during KSHV reactivation (TPA + caspase inhibitor) using inhibitors like SP600125 and subsequently assess whether this blockade reduces the proportion of IFNb+ cells.

    Significance

    The study presents a valuable dataset and serves as a crucial starting point for understanding cellular heterogeneity, particularly regarding the known concept of IRF+NFkB in IFNb production. While this mechanism isn't novel (10.1074/jbc.273.5.2714), the authors demonstrated the difference in activation in a cellular level. This finding can be the basis of future research utilizing more physiologically relevant models, such as primary cells or tissues, to identify factors contributing to varying cellular responses.

    However, the authors acknowledge that these findings should be interpreted with caution and require further validation through additional studies across different models and viral infections. This research will be particularly relevant to those in basic research seeking to deepen their understanding of the dynamic differences in innate immune responses and viral infections.

  3. Review Commons

    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

    In the manuscript "Interferon-β induction heterogeneity during KSHV infection is correlated to expression and activation of enhanceosome transcription factors other than IRF3", Kaku and Gaglia address a long-standing question in the initiation of host antiviral responses: what drives the heterogeneity in the initiation of IFN responses within a presumably homogenous population of cells. In this study, the authors focus on host factors that contribute to the heterogeneity in IFN induction. They use a KSHV lytic reactivation model (TPA + Caspase inhibitor treated BC-3 cells) and FACS-based reporter assays to enhance the resolution in the detection of molecular drivers of "first responder" cells that make IFN. They find that IRF3 activation alone does not predict IFN expression; rather, the expression of ATF2 and RelA is predictive of IFN-β induction. The authors carefully control for off-target effects of TPA treatment in BJAB cells and paracrine signaling through the inclusion of IFN-neutralizing antibodies. Overall, the manuscript is well-written and easy to follow, and the data compellingly support their conclusion that cell-specific transcription factor activity limits IFN production to cell subsets. Demonstrating coordinated occupancy or functional interplay of these factors would increase confidence in the proposed model and broaden the impact for readers interested in virology, immunology, and transcriptional regulation.

    Comments:

    1. While the study presents intriguing evidence for AP-1 involvement in regulating IFN-β responses, the reliance on total c-Jun levels as a readout is limiting. Because c-Jun activity depends on phosphorylation and promoter binding, additional experiments (i.e., phospho-c-Jun analysis or ChIP at the IFNB1 promoter) would strengthen the link between AP-1 activity and the observed reporter outcomes.
    2. The data presented demonstrating that Serine 386 phosphorylation does not distinguish first responder cells is strong. Including complementary data on Ser396 phosphorylation would strengthen the conclusion, as this well-established activation marker is readily detectable with available reagents and would help confirm that the potentiation of IRF3 activity is not the driver of the observed heterogeneity.

    Minor Comments:

    1. Consider updating the title to more directly reflect the findings (e.g. "Interferon-β induction heterogeneity during KSHV infection is correlated to expression of ATF2 and RelA".
    2. To ease the interpretation of data, indicate what the black and white circles indicate in the figure legends.
    3. The authors predominantly rely on the IE gene ORF50 as a marker of KSHV reactivation and show no differences in expression between first responder cells and those that don't. Measurement of early and late genes across TdTomato-positive and negative cells would rule out potential differences in progression through reactivation that might influence IFN production.
    4. The data in Figure 5D (quantified in E and F) show a compelling trend. This could be further clarified by plotting a trend line that connects the results of independent experiments, rather than only showing individual data points. Such visualization would make the consistency of the observed trend across experiments more apparent.

    Significance

    This study addresses an important and long-stading question in the heterogeneity of cellular responses to stressors, such as viruses. The study is well designed and presented, making it appealling to a broad audience interested in virology, immunology, cell biology, and gene transcription.

  4. Review Commons

    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

    The manuscript discusses why only a small proportion of KSHV infected cells produce high level of IFN during lytic reactivation in the presence of caspase inhibition, highlighting cellular heterogeneity as a key factor in innate immune regulation in KSHV infection. Using a dt-TOMATO reporter fused to the IFN promoter, the author generated a stable cell line in BC3 cells (a primary effusion lymphoma line). The authors observed that while IRF3 and TBK1 were activated in nearly all infected cells, only those with both high baseline levels of the AP-1 component ATF2 and activated NF-κB (phosphorylated RelA) produced robust FN. These findings suggest that AP-1 and NF-B, rather than IRF3, are the limiting factors for IFN induction in individual cells.

    Overall the findings are interesting and important. While there remain many unknowns, such as why RelA is activated in only a subset of cells, this manuscript takes us one step close to determining how IFN is ultimately induced in KSHV infection.

    One experiment that may provide some insight into the selective RelA activation is to quantify viral genomes within the high and low IFN producing cells. Perhaps, the genome as a PAMP, is more abundant in the inducing cells.

    Significance

    The study makes a conceptual advance by identifying transcription factors as the limiting determinant of IFN-β induction in KSHV-infected cells, highlighting how innate immune responses are regulated during herpesvirus infection and how the regulation influences viral persistence and immune evasion.

    The above findings will be of important to researchers studying herpesvirus biology, innate immunity (IFN signaling), and host-pathogen interaction.

  5. Version published to 10.1101/2024.12.22.629998 on bioRxiv