Epigenetic remodeling by vitamin C potentiates plasma cell differentiation
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Curated by eLife
Evaluation Summary:
This manuscript described a role of Vitamin C in promoting plasma cell differentiation by remodeling the epigenome via TET (Ten Eleven Translation) family proteins. Overall, most of experiments are properly executed, controlled and presented. This paper will be of interest to scientists in molecular immunologists, particularly those involved in of epigenetic mechanisms of B cell differentiation to plasma cells.
(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. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Ascorbate (vitamin C) is an essential micronutrient in humans. The severe chronic deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. From a micronutrient analysis, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique as other antioxidants failed to promote plasma cell differentiation. Ascorbate is especially critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. As a cofactor for epigenetic enzymes, ascorbate facilitates TET2/3-mediated DNA modification and demethylation of multiple elements at the Prdm1 locus. DNA demethylation augments STAT3 association at the Prdm1 promoter and a downstream enhancer, thus ensuring efficient gene expression and plasma cell differentiation. The results suggest that an adequate level of ascorbate is required for antibody response and highlight how micronutrients may regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.
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Author Response
Reviewer #1 (Public Review):
In this paper the authors find that vitamin C (VC) enhances the differentiation of B cells to plasma cells (PC) in an in vitro culture system and link the treatment regimen to changes in the DNA methylation pattern changes associated with B cell differentiation. The work generally supports the conclusions. The differentiation of B cells to PC is critical to the induction of adaptive immunity to infection and vaccination.
Strengths: 1) The major strength of the paper is the observation that VC greatly enhances plasma cell formation in the culture assay. 2) Because they have a two step differentiation process, the authors were able to narrow down the important point of VC action on the first step as IL-21 signaling did not change. 3) The authors focused the rest of the studies on the …
Author Response
Reviewer #1 (Public Review):
In this paper the authors find that vitamin C (VC) enhances the differentiation of B cells to plasma cells (PC) in an in vitro culture system and link the treatment regimen to changes in the DNA methylation pattern changes associated with B cell differentiation. The work generally supports the conclusions. The differentiation of B cells to PC is critical to the induction of adaptive immunity to infection and vaccination.
Strengths: 1) The major strength of the paper is the observation that VC greatly enhances plasma cell formation in the culture assay. 2) Because they have a two step differentiation process, the authors were able to narrow down the important point of VC action on the first step as IL-21 signaling did not change. 3) The authors focused the rest of the studies on the actions of the TET2/3 proteins, which connects the iron pathway as a cofactor for the TET enzymes and antioxidation nutrients such as VC. 4) The authors use a relatively novel chemistry to assess 5hmC levels. 5) The data appear to have been rigorously collected with an appropriate number of samples.
We would like to thank you for the comments.
Weaknesses: 1) The direct connection between IL-21 STAT3 signaling and the E58 region of the Prdm1 gene is not shown, but rather inferred from previous work in T cells. Because this is "the connection," they should attempt to show this by ChIP in their system. It should be possible as the experiments are in vitro and lots of cells can be generated with a high proportion differentiating cells in the culture.
We appreciate the Reviewer’s suggestions, which allowed us to identify another VC-regulated element E27 at the Prdm1 locus in B cells.
- We were unable to completely reproduce the previous T cell STAT3 ChIP-seq by Kwon et al., 2009 (Dr. Warren Leonard’s lab) due to the discontinuation of the antibody (Santa Cruz). Therefore, we have tested four anti-STAT3 antibodies (Cell Signaling Technology rabbit monoclonal #4904, #12640, #9145; Millipore rabbit polyclonal #06-596), and found that only the rabbit mAb #9145 anti-phospho-Stat3 (Tyr705) from CST generated a specific signal for ChIP, which was quantified by qPCR using an IL-21-induced positive control Mcl1.
Surprisingly, the ChIP-seq result showed that the STAT3 binding patterns differ between B cells and the previously published data from T cells (New Fig. 8D and S8-1B). The difference may likely be due to cell types, culture conditions, and the source of antibodies. Nonetheless, our STAT3 ChIP-seq data showed that VC could enhance STAT3 binding at the Prdm1 promoter and E27, a previously identified enhancer with a functional STAT3 motif at +27kb (New Fig. 8D)(Kwon et al., 2009). In order to prove that the DNA modification of this element was indeed regulated by VC, we analyzed the DNA methylation using bisulfite nanopore sequencing and showed that VC induced DNA demethylation at E27 (NEW Fig. 9A). Importantly, both VC-enhanced DNA methylation (NEW Fig. 9B) and STAT3 binding (NEW Fig. 9C) at E27 were abolished in the Tet2/3-deficient B cells. Our data strongly corroborate with the previously identified STAT3 binding site at E27 by Kwon et al., where they showed that the STAT3 motif is responsible for IL-21-induced Prdm1 expression. Our results further demonstrated that E27 is regulated by DNA methylation and is thus sensitive to the status of VC.
- From the 5hmC DNA dot blot, it is difficult to make the interpretation that there is an increase in activity of the TETs during the process as the VC samples look like naïve cells and there is a clear loss of 5hmC in the Mock treated samples that stays relatively the same during the differentiation process. A better description of the logic and new sites that go from 5mC to 5hmC is needed.
We have now included additional explanation in the text and a new supplementary figure (NEW Fig. S7-1).
Line 264 TET enzymes oxidize 5mC into 5hmC, a stable epigenetic medication and an intermediate for DNA demethylation (Fig. S7-1A)40,56,64. To confirm that VC indeed enhances TET activity, we used DNA dot blot to semi-quantitatively measure the level of 5hmC in B cells. Naïve B cells had the highest density of 5hmC compared with B cells from day four or day seven culture (Fig. 7A). The decreased 5hmC is consistent with the passive dilution of 5hmC after cell divisions, as the 5hmC modification pattern is not replicated on the newly synthesized DNA (Fig. S7-1B-C).
Reviewer #3 (Public Review):
This paper titled "Epigenetic remodeling by Vit C potentiates the differentiation of mouse and human plasma cells" is a very interesting paper with novel findings and mechanisms of Vit C in plasma cell differentiation. They elegantly show that Vit C enhances plasma cell differentiation and that the mechanisms involve TET activity and DNA demethylation. Their current model supports plasma cell differentiation of IgM, IgE and IgG1 in mouse which is a type 2 immune response model. If VIt C can be shown in a type 1 immune model, it would important. Then, these data may help to support Linus Pauling's claims that Vit C prevents and alleviates the common cold if the model can be applied broadly.
We sincerely appreciate and are thankful for the Reviewer’s comments. We hope our study will provide insight into how VC may contribute to a proper immune response.
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Evaluation Summary:
This manuscript described a role of Vitamin C in promoting plasma cell differentiation by remodeling the epigenome via TET (Ten Eleven Translation) family proteins. Overall, most of experiments are properly executed, controlled and presented. This paper will be of interest to scientists in molecular immunologists, particularly those involved in of epigenetic mechanisms of B cell differentiation to plasma cells.
(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. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper the authors find that vitamin C (VC) enhances the differentiation of B cells to plasma cells (PC) in an in vitro culture system and link the treatment regimen to changes in the DNA methylation pattern changes associated with B cell differentiation. The work generally supports the conclusions. The differentiation of B cells to PC is critical to the induction of adaptive immunity to infection and vaccination.
Strengths: 1) The major strength of the paper is the observation that VC greatly enhances plasma cell formation in the culture assay. 2) Because they have a two step differentiation process, the authors were able to narrow down the important point of VC action on the first step as IL-21 signaling did not change. 3) The authors focused the rest of the studies on the actions of the TET2/3 …
Reviewer #1 (Public Review):
In this paper the authors find that vitamin C (VC) enhances the differentiation of B cells to plasma cells (PC) in an in vitro culture system and link the treatment regimen to changes in the DNA methylation pattern changes associated with B cell differentiation. The work generally supports the conclusions. The differentiation of B cells to PC is critical to the induction of adaptive immunity to infection and vaccination.
Strengths: 1) The major strength of the paper is the observation that VC greatly enhances plasma cell formation in the culture assay. 2) Because they have a two step differentiation process, the authors were able to narrow down the important point of VC action on the first step as IL-21 signaling did not change. 3) The authors focused the rest of the studies on the actions of the TET2/3 proteins, which connects the iron pathway as a cofactor for the TET enzymes and antioxidation nutrients such as VC. 4) The authors use a relatively novel chemistry to assess 5hmC levels. 5) The data appear to have been rigorously collected with an appropriate number of samples.
Weaknesses: 1) The direct connection between IL-21 STAT3 signaling and the E58 region of the Prdm1 gene is not shown, but rather inferred from previous work in T cells. Because this is "the connection," they should attempt to show this by ChIP in their system. It should be possible as the experiments are in vitro and lots of cells can be generated with a high proportion differentiating cells in the culture. 2) From the 5hmC DNA dot blot, it is difficult to make the interpretation that there is an increase in activity of the TETs during the process as the VC samples look like naïve cells and there is a clear loss of 5hmC in the Mock treated samples that stays relatively the same during the differentiation process. A better description of the logic and new sites that go from 5mC to 5hmC is needed.
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Reviewer #2 (Public Review):
Heng-Yi Chen, Chan-Wang Jerry Lio and collaborators address the impact of Ascorbate (Vit C) on plasma cell differentiation, by segregating Vit C's antioxidant activity from Vit C epigenetic activity, i.e., as provider of the Tet2/Tet3 co-factor Fe++. The data presented convincingly show that Vit C promotes B cell differentiation to plasma cell by boosting Tet2/Tet3 activation. Activated Tet2/Tet3 actively demethylate selected genomic regions, including and importantly, the Prdm1 locus, the gene encoding Blimp-1, the master transcription factor of plasma cell differentiation.
Equally convincing data are provided that Vit C does not boost B cells differentiation to plasma cells as antioxidant, that is, Tet2/Tet3 activity on the Prdm1 locus is independent of Vit C general antioxidant activity. Indeed, other …
Reviewer #2 (Public Review):
Heng-Yi Chen, Chan-Wang Jerry Lio and collaborators address the impact of Ascorbate (Vit C) on plasma cell differentiation, by segregating Vit C's antioxidant activity from Vit C epigenetic activity, i.e., as provider of the Tet2/Tet3 co-factor Fe++. The data presented convincingly show that Vit C promotes B cell differentiation to plasma cell by boosting Tet2/Tet3 activation. Activated Tet2/Tet3 actively demethylate selected genomic regions, including and importantly, the Prdm1 locus, the gene encoding Blimp-1, the master transcription factor of plasma cell differentiation.
Equally convincing data are provided that Vit C does not boost B cells differentiation to plasma cells as antioxidant, that is, Tet2/Tet3 activity on the Prdm1 locus is independent of Vit C general antioxidant activity. Indeed, other antioxidants, including vitamin E, reduced glutathione, NAC, and 2-mercaptoethanol did not recapitulate the effect of Vit C. And the basal levels of oxidative stress were low in B cells regardless of Vit C, consistent with the that Vit C exerts its activity on other cell processes (Tet2/Tet3 activation).
The last point is important as one of the new information provided by this work over the recent paper by Qi, T. et al. (Ascorbic Acid Promotes Plasma Cell Differentiation through Enhancing TET2/3-Mediated DNA Demethylation - Cell Rep 33, 108452, 903, 2020), showing that Tet2/Tet3 critically boost plasma cell differentiation by inducing expression of Blimp1, through active demethylation of the Prdm1 locus, but not addressing a potential role of Vit C as antioxidant in plasma cell differentiation.
Other novelties of this work include:
The demonstration that the main effect of Vit C occurs during the initial activation stage of stimulation of B cells in their process of undergoing plasma cell differentiation, prior to the subsequent stage as promoted by IL-21. This Vit C effect cannot be replaced by other antioxidants.
The finding that Vit C has a profound effect on the epigenome, including the genome-wide distribution of 5hmC. Vit C significantly increased the total 5hmC deposited by Tet2/Tet3, being responsible for appearance of at least 10,000 de novo 5hmC peaks induced in conjunction with B cell activation.
Finally, while the Qi, T. et al. 2020 paper provided in vivo data, the current work details the molecular mechanisms of how Vit C boosts plasma cell differentiation, including the demonstration by genome-wide profiling of 5hmC of several "ascorbate responsive elements" (EARs, defined as regions with gained 5hmC after Vit C treatment) in the Prdm1 locus, including a potential novel element E58 (element located at +58kb from the start of Prdm1) that might be sensitive to the methylation status.
All in all, the above multiple novel points, the intrinsic quality of the data, their clear presentation together with the overall dissection of the sequential Tet2/Tet3-driven B cell differentiation steps toward plasma cell temper the the possible perception the the work may just be incremental over the in vivo paper by Qi, T. et al. 2020.
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Reviewer #3 (Public Review):
This paper titled "Epigenetic remodeling by Vit C potentiates the differentiation of mouse and human plasma cells" is a very interesting paper with novel findings and mechanisms of Vit C in plasma cell differentiation. They elegantly show that Vit C enhances plasma cell differentiation and that the mechanisms involve TET activity and DNA demethylation. Their current model supports plasma cell differentiation of IgM, IgE and IgG1 in mouse which is a type 2 immune response model. If VIt C can be shown in a type 1 immune model, it would important. Then, these data may help to support Linus Pauling's claims that Vit C prevents and alleviates the common cold if the model can be applied broadly.
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