Crosstalk between the RNA-binding proteins Regnase-1 and -3 shapes mast cell survival and cytokine expression

  1. Marian Bataclan
  2. Cristina Leoni
  3. Simone G Moro
  4. Matteo Pecoraro
  5. Elaine H Wong
  6. Vigo Heissmeyer
  7. Silvia Monticelli

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Abstract

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, while both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon mast cell activation, with Tnf being a primary target of both proteins. In mast cells, Regnase-3 directly interacts with Regnase-1 and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

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    Reply to the reviewers

    Please see attached point-by-point response file, it contains essential figures and formatting that cannot be pasted here.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Bataclan et al. provide extensive in vitro work on Regnase1/3 function in mast cells. They make use of in vitro differentiated mast cells from bone marrow (BMMC) and in vitro cultures from primary peritoneal mast cells from mice. They show robustly Reg1/3 upregulation by transcript and protein upon activation stimulation in these cultured mast cells. Knockdown and CRISPR editing of Reg1/3 show enhanced inflammatory signaling. TNF is identified as a direct target of Reg1/3 nuclease activity. Reg3 is implicated as a negative regulator of Reg1. Overall, this work highlights Reg1/3 as novel players in mast cell biology that control inflammatory signaling including TNF.

    Major comments:

    • The result section requires more consistent & sufficient information on the assays (in vitro, ex vivo, species, etc.) particularly for the meta-analysis in the beginning. Relatedly, can the authors assume cross-species conservation of Reg1/3 in mast cells across mammals to make general conclusions on the functions or should this be limited mostly to mouse observations? It would be helpful to mention the species studied in the abstract.
    • Reg1 protein levels appear to be more stable than Reg3 protein levels (mirroring the transcript) Fig1a+c. Is the half-life of Reg3 shorter than Reg1? What is functional impact of Reg1 regulation at the transcriptional level considering the protein half-lives?
    • MFI comparisons can only be used on unimodal populations (not bimodal i.e. Fig3b). Also, contour flow plots should show outliers.
    • Is TNF functionally secreted from WT vs KD/KO mast cells under these in vitro conditions? This is not shown and seems quite an important aspect given the focus on TNF regulation (other secreted factors could also be considered). Degranulation appears to be lower in Fig5g. Does Reg1 overexpression reduces TNF expression and secretion?
    • Catalytic variants of Reg1/3 appear to be only tested in WT cells. Wouldn't it be worthwhile to test these in KO cells? The absolute protein levels of ectopic and endogenous are not entirely clear and only shown for WT Reg3 in Fig4b.
    • Fig2b shows upregulation of Reg1 transcript is not enhanced upon Reg3 knockdown, but it is enhanced in Fig4a? How are these assays different?
    • The authors implicate Reg3 to regulate Reg1 by transcript. What is the role of the protein interaction between Reg1 and 3? Why is the protein interaction not seen in the IF experiments? Would other cell systems (maybe mouse) be more suitable.
    • Cell death increases after Reg1/3 knockdowns; can this be rescued with Reg1/3 reinstation? Is the protein level of Reg1 important for this; can this be dosed? Are the assays for cell death/proliferation done in comparison to unstimulated resting cells, cultured conditions and IgE stimulated mast cells? Minor point: A single assay of cell death is sufficient in the main figures; same for proliferation assays.
    • It is not entirely clear how the RNA-seq data with CRISPR KO in Fig6 is different than the nanostring data with Knockdowns shown earlier in Fig2? Were unstimulated cells not used as a control before? A floxed mouse model for Reg1 is introduced at the end, but would have been useful for many assays. OPTIONAL: are in vivo experiments of interest to confirm the findings? The conclusions that Reg1/3 regulates physiological mast cell responses might be a reach otherwise.

    Minor comments:

    • Some of the Knockdown and CRISPR validation main figures are redundant and might be better suited for the supplement.
    • The MW in the western blot requires lines to indicate the exact location of the ladder.
    • Fig2 title refers as loss of Reg1/3 using siRNA. The term is typically used in the context of genetic ablation and not for knockdowns.
    • Gene locus figures of Reg1/3 are confusing why are only E3 and E6 shown and not all Exons (Fig2a et al)?
    • Are biological replicates or technical replicates used in Fig1?
    • Is a student's t test the appropriate statistical analysis for most of the analyses?
    • An explanation why nanostring and RNA-seq are both used would be helpful.

    Significance

    Bataclan et al investigate Reg1/3 function in mast cells, that are of interest to elucidate the regulation of mast cell effector functions. Reg1/3 were identified as novel regulators of murine mast cells. Independent and complementary use of Knockdowns and CRISPR deletions provide robust data. Also, the use of two independent mast cell populations and different stimuli is of interest, albeit they are not used in every assay. Altogether, the in vitro data are robust and rigorous. On the flipside, in vivo data is not provided. The translational impact would be higher if findings are tested in in vivo models including preclinical applications (as discussed in text). Mouse studies are often indicative of mechanisms in higher mammals, but it would help to lay this out more clearly. Therefore, the connection to humans is less clear. The type of work presented here would be best described as basic research. This review has been assessed with the following expertise: mouse/human immunology, mouse models, immune cell signaling, immune effector functions, flow cytometry.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Bataclan et al. studied the role of Regnase-1 and -3 in the mast cell (MC) function in vitro. Using siRNA, CRISPR-mediated depletion, and overexpression models in, mainly, IL-3-cultured bone-marrow-derived mast cells, they beautifully demonstrated that Regnase-1 and, to a lesser extent, Regnase-3 suppressed the expression of TNF in MCs in response to IgE-stimulation. Regnase-1 also had roles in MC survival and IgE-stimulation-induced degranulation. Regnase-3 seems to have mixed effects on MC functions as the protein's primary targets include both TNF and Regnase-1 mRNAs.

    Major comments:

    1.      The authors mainly used BMMCs generated by four weeks of cultivation of bone marrow cells with IL-3; at this point, MCs are considered "less mature". Could the authors reproduce the primary data (the role of Regnase-1 on TNF, cell survival, and degranulation) even if they used more mature MCs (e.g., extended cultivation with IL-3 (6-7 weeks))?

    2.      TNF mRNA is not considered a primary target for Regnase-1 in other cell types, such as macrophages or fibroblasts. Although this reviewer does not doubt that the TNF expression level is controlled by Regnase-1 in MCs, more evidence is needed to conclude that TNF is a "primary target" for Regnases. They can determine the stability of "endogenous" TNF mRNA in Regnase-depleted cells, as in Figure 4d.

    3.      The role of Regnase-1 on the MC degranulation is less pronounced. Could the authors show the same result using another assay, such as a FACS-based assay excluding dead or dying cells?

    4.      Have the authors had a chance to look into which protease(s) cleaved Regnase-1 in IgE-stimulated MCs?

    Significance

    General assessment:

    This study investigated the role of Regnase-1 and -3 in the mast cell (MC) function in vitro. The strength of the study is that they used several methods to manipulate the expression levels of Regnases in the cells (siRNA, CRISPR, and Regnase-1 overexpression) and obtained consistent results. Although the study is well designed and the results are beautiful, the BMMCs the authors used are less mature MCs and do not represent the cells in the mammalian body well. Also, this study only focused on in vitro mouse-derived MCs, and human MCs or in vivo roles of MC Regnases were not studied.

    Advance:

    The role of Regnase-1 in several cell types has already been shown, including the populations involved in type-2 immunity (Th2 and ILC2). However, this study might be the first to investigate the role of Regnases in MCs. The roles of Regnases in MCs (control of mRNA stability through the RNase activity) presented are in line with the previous studies.

    Audience:

    The primary audience of this study may be basic researchers studying type-2 immunity or MC biology. Because MCs are an essential cell population in several allergic disorders, some clinicians who care for allergic patients might also be interested in this study. However, main audiences may be relatively limited to specific fields like immunology and allergology.

    This reviewer's main field of expertise is basic research in immunology and allergology. More specific keywords are MCs, ILC2, IgE, type-2 cytokines, and mouse models.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Mast cells play a role in exerting effector functions in the immune response. However, the functioning of RNA-binding proteins (RBPs) in the mast cells is not well understood. The authors focused on the Regnase family of RNA-degrading enzymes and worked towards unraveling their functions. Upon activating mast cells, the authors observed a significant induction of Regnase-3 among RBPs. Furthermore, they found that Regnase-3 directly controls the well-known Regnase-1, revealing its role in regulating homeostasis and inflammatory responses in mast cells.

    Major comments:

    This experiment has been meticulously designed, and the reliability of the presented data is quite high. While the observations are specific to mast cells, the insights gained could provide valuable information about the interrelation within the Regnase family across other immune cell types. However, there are three main concerns raised by the reviewer:

    The first concern revolves around the use of the IVT system for the expression of Regnase-1 and Regnase-3. Is there evidence confirming the expression of Regnase-3 as a full-length protein, as shown in Figures 3g and 3h? The APC-HA signal by FACS could be positive even for degradation products alone. Assuming Regnase-3 is not expressed in its full-length, it might lead to results similar to the control. Given the larger molecular weight of Regnase-3 compared to Regnase-1, it is crucial to demonstrate sufficient expression through IVT. In particular, the Western blot data for Regnase-3 in some cases confirm the full length of the product, while in other cases more degradation products appear.

    The second concern arises from the co-immunoprecipitation experiment in Figure 4i. While the experiment detects Regnase-1 co-precipitating with Regnase-3, the reverse-precipitating Regnase-1 with Regnase-3 shows a signal comparable to IgG, indicating background noise. Further improvement is needed in this aspect.Is it possible that your antibody against Regnase-1 also binds to Regnase-3?

    The third concern is related to the evaluation of Regnase-1 degranulation in Figure 5g, where there appears to be some variability. Including Regnase-3 and conducting mutual evaluations could enhance the reliability of the results, possibly addressing this variability.

    Minor comments:

    Figure 6e-g also seems to vary, and the effects on cell death and cell proliferation tend to be somewhat milder.

    Certainly, their IF images (Figure S2 and Figure S5) suggests that Regnase-1 and Regnase-3 have no or only a weak interaction.

    Is the input to the immunoprecipitation from whole cell lysate or is it cleared with a low-speed (or high-speed) centrifugation?

    Significance

    The following aspects are important: Understanding the regulatory mechanisms mediated by RNA-binding proteins (RBPs) has gained significant attention in recent years. While it is inferred that RBPs control specific RNAs, many uncertainties remain about how they function in various RNA metabolism processes. The Regnase family is known to operate within the mechanism of RNA stability. While lower organisms have only one type, mammals have evolved to have four types. Understanding the implications of this family expansion is highly valuable, shedding light on how the RNA within our body's cells is regulated.

    • Advance: In this study, a strength lies in the meticulous examination of the relationship between Regnase-1 and Regnase-3 by handling them similarly in the context of mast cells. However, because of the multifaceted effects of Regnase-1 on cell proliferation, cell death, and the cell cycle, significant progress in understanding it has yet to be made. Going forward, the replication of immune responses in mast cells at the animal level holds the potential to further deepen our comprehension of RBPs through the Regnase family. This study complements two previous investigations on Regnase-3 (PMID: 34215755, 31126966) while specifically focusing on mast cells. The findings align with the prevailing perspective that emphasizes the significance of Regnase-1 among the Regnase family.
    • Audience: Basic research
    • Immunology, Molecular Biology, Genetic Engineering
  5. Version published to 10.1101/2024.01.24.577016v1 on bioRxiv