The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells
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Evaluation Summary:
The possibility that local or systemic hypoxia regulates aspects of the immune system has attracted widespread interest over the last decade or more. Previous work has implicated regulatory T cells in at least some of these responses, and has demonstrated that over-activation of a specific hypoxia inducible factor (HIF) isoform, HIF-2alpha has the potential to driven pro-inflammatory lymphoproliferative responses characterized by defective regulatory T cells. The current work demonstrates that genetic activation of these hypoxia-signalling pathways that is restricted to the regulatory T cell lineage is sufficient to drive this type of immune activation. The work is important since it provides a focus for study of the mechanism, for which the authors make a proposal based on mis-localization of regulatory T cells. It is also important in focussing a key questions (requiring further study) as to whether physiological or pathological hypoxia, specifically affecting these cells, will drive such a response and/or whether the lymphoproliferative phenotype could be affected adversely or beneficially by agents that are being used to upregulate or downregulate hypoxia-signalling pathways in other settings.
(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 #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
The oxygen sensor prolyl hydroxylase domain 2 (PHD2) plays an important role in cell hypoxia adaptation by regulating the stability of HIF proteins (HIF1α and HIF2α) in numerous cell types, including T lymphocytes. The role of oxygen sensor on immune cells, particularly on regulatory T cell (Treg) function, has not been fully elucidated. The purpose of our study was to evaluate the role of PHD2 in the regulation of Treg phenotype and function. We demonstrate herein that selective ablation of PHD2 expression in Treg (PHD2 ΔTreg mice) leads to a spontaneous systemic inflammatory syndrome, as evidenced by weight loss, development of a rectal prolapse, splenomegaly, shortening of the colon, and elevated expression of IFN-γ in the mesenteric lymph nodes, intestine, and spleen. PHD2 deficiency in Tregs led to an increased number of activated CD4 conventional T cells expressing a Th1-like effector phenotype. Concomitantly, the expression of innate-type cytokines such as Il1b , Il12a , Il12b, and Tnfa was found to be elevated in peripheral (gut) tissues and spleen. PHD2 ΔTreg mice also displayed an enhanced sensitivity to dextran sodium sulfate-induced colitis and toxoplasmosis, suggesting that PHD2-deficient Tregs did not efficiently control inflammatory response in vivo, particularly those characterized by IFN-γ production. Further analysis revealed that Treg dysregulation was largely prevented in PHD2-HIF2α (PHD2-HIF2α ΔTreg mice), but not in PHD2-HIF1α (PHD2-HIF1α ΔTreg mice) double KOs, suggesting an important and possibly selective role of the PHD2-HIF2α axis in the control of Treg function. Finally, the transcriptomic analysis of PHD2-deficient Tregs identified the STAT1 pathway as a target of the PHD2-HIF2α axis in regulatory T cell phenotype and in vivo function.
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Author Response
Reviewer #1 (Public Review):
In this paper the authors use a conditional knockout strategy to assess the effects of deletion of the dominant oxygen-sensing hypoxia-inducible factor (HIF) hydroxylase enzyme, prolyl hydroxylase 2 (Phd2) restricted to the regulatory T cell (Treg) lineage. They use a well-established Foxp3-driven Cre recombinase allele. Phd2 is thus silenced in cells that have expressed or continue to express Foxp3 from the time this transcription factor, which is essential for Treg development and function, first occurs. They show that this approach leads to a change in Treg behaviour resulting in loss of some aspects of regulatory function and development of a Th1-like phenotype by the Foxp3 expressing cells. Effects are in general reversed when HIF-2 is silenced alongside Phd2, and may be amplified by …
Author Response
Reviewer #1 (Public Review):
In this paper the authors use a conditional knockout strategy to assess the effects of deletion of the dominant oxygen-sensing hypoxia-inducible factor (HIF) hydroxylase enzyme, prolyl hydroxylase 2 (Phd2) restricted to the regulatory T cell (Treg) lineage. They use a well-established Foxp3-driven Cre recombinase allele. Phd2 is thus silenced in cells that have expressed or continue to express Foxp3 from the time this transcription factor, which is essential for Treg development and function, first occurs. They show that this approach leads to a change in Treg behaviour resulting in loss of some aspects of regulatory function and development of a Th1-like phenotype by the Foxp3 expressing cells. Effects are in general reversed when HIF-2 is silenced alongside Phd2, and may be amplified by simultaneous silencing of the HIF-1 isoform.
The findings overlap with those reported following generalised silencing of Phd2 and following adoptive transfer of Treg in which Phd2-silencing is induced (Yamamoto et al., 2019) and are broadly compatible with those reported following a similarly Treg-restricted knockout of the von Hippel-Lindau gene (the recognition component of the E2-ubiquitin ligase that targets HIF-alpha chains that have been modified by Phd2) (Lee et al., 2015) but the results reported also differ significantly from these earlier reports in a number of intriguing respects which I feel warrant further discussion and ultimately investigation.
The Introduction is in general informative and well written but it is a shame that it does not contain more discussion of the current state of knowledge of the interplay between HIF signalling and Treg function. This would provide a platform for a more detailed and scholarly discussion of the similarities and differences between this work and existing literature in the Discussion, where existing papers are currently described rather briefly. The introduction contains the statement 'Further complexity in this pathway has been provided by the identification of additional, non-HIF-related, PHD substrates, suggesting a role of proline hydroxylation in other settings requiring oxygen-dependent regulation', citing a single reference. This does not really represent the complex balance of arguments across the literature about non-HIF substrates for the HIF hydroxylase enzymes.
The conclusions of this paper are mostly well supported by data, but some aspects need to be clarified and extended.
We sincerely apologize for our apparent lack of recognition of previous work performed by other colleagues active in this field. We have now modified the Introduction section, to provide a better, yet concise, overview of the current knowledge of hypoxia signalling in regulatory T cell biology.
A central issue for any conditional knock-out strategy is whether the intended tissue restriction is successfully achieved. The authors acknowledge that some issues have been reported with the Cre-recombinase allele they use. They, however, show the expected restriction to cells of the Treg lineage in two of the lymphoid tissues under investigation (spleen and mesenteric lymph node - Supplementary figure 1b) but do not show similar results for other tissues. Some concerns arise because in Figure 8b YFP (which is expressed alongside the Cre-recombinase) is visible in what appears to be the endothelium of the spleen. Additionally, the spleen sections illustrated show convincing splenomegaly in the Phd2-deficient Treg mice but expansion of the red pulp appears to be at least as prominent as any changes that might have occurred in the white pulp. Furthermore, the gross changes in abdominal appearances described as a 'hemorrhagic abdomen' (Figure 1c) include a more plethoric abdominal wall, prominent intestinal blood vessels and a much darker, and perhaps enlarged, liver compared with the control animal. These appearances might result from increased angiogenesis and / or erythropoiesis, neither of which would be expected to result from Treg lineage restricted Phd2 knockout but are known to occur with Phd2 ablation in other tissues. If there is convincing evidence of haemorrhage it would be nice to see this more obviously displayed macro- or, perhaps better still, microscopically.
We thank the reviewer for this comment. We have now provided a better description of the haematological status of these mice, in which an elevated haematocrit and increased vascular permeability has been observed (now depicted in supplemental Figure 2). As suggested, we found indeed minimal, yet sizable expression of the Cre recombinase (as judged by YFP expression) in CD45-negative, non-lymphoid cells in all organs examined (as now depicted in supplemental Figure 9). Finally, none of the organs examined displayed an increased expression of erythropoietin (as judged by a sensitive qPCR assay, data not shown), a likely candidate for the haematological abnormalities observed in these mice. The mechanism underlying the apparent extramedullary erythropoiesis occurring in these mice remains therefore to be established. Noteworthy however, an additional experiment performed following a suggestion from one of the reviewers (see Figure 3 and our response 23), strongly suggests that PHD2 affects the Treg phenotype in a cell autonomous fashion. We do however acknowledge that the tissue abnormalities preclude any firm conclusion related to the positioning of Tregs within the spleen and have therefore deleted this section from the manuscript and adapted our conclusion consequently.
Given that the Cre-recombinase allele used is expressed through the endogenous Foxp3 locus which is located on the X-chromosome and thus subject to random inactivation in the cells of females it is important that the sex of animals used in the experiments is specified.
This has now been done in the Figure legends
Experiments show alterations in Phd2-deficient Treg mice compared with control mice in homeostatic proliferation in a lymphopenic environment (Figure 3), the induction of colitis by DSS colitis (Figure 4) and the response to Toxoplasma gondii infection (Figure 4). Given the time courses these effects are likely to be real but interpretation is complicated by the spontaneous effects on the colon of Phd2-deficient Treg mice reported in Figure 1d and e. Given the wide general importance of interferon-gamma in immune / inflammatory responses I am not sure how much weight to place on the observation that concurrent interferon-gamma knockout results in loss of the Phd2-deficient Treg mice pro-inflammatory phenotype (Figure S3). No differences are seen in an in vivo model in which inflammation is induced by injection of anti-CD3 antibodies (Figure S2).
Although the point is well taken, we felt it was important to perform a few experiments to illustrate the specificity of the inflammatory syndrome observed in these mice. We acknowledge the fact that the effect of concurrent loss of interferon-gamma on the phenotype of PHD2ΔTregs could have been anticipated. Additionnaly, we also think that the fact that these mice retain the same sensitivity to a “Th17-dominated” inflammatory response (also leading to a loss of weight) strengthens one of the messages of the manuscript, i.e. that loss of PHD2 expression affects Treg function in a selective, Th1-oriented fashion.
An important conceptual difference between the interpretation of results reported here and those reported by Yamamoto et al. is that the 'Phd2-deficient Treg' purified here do not show a change in regulatory function in vitro whereas those used by Yamamoto et al. failed to act normally as regulatory cells. It is unclear whether this is due to differences in the way proliferation was stimulated, the cell purification strategies used (YFP+ in the current work; CD4+;CD25+ in Yamamoto et al.), the silencing of Phd2 (by knockout throughout development here versus through an inducible-shRNA only in mature cells in Yamamoto et al.), some other feature of the experiments (e.g. the use of feeder cells) or whether a difference would be revealed by more extensive titration. The result reported here is somewhat surprising given the presence of a Th1-like immunophenotype in the cells used in these in vitro suppression assays, which at face value might mean that this immunophenotype is not responsible for changes in their regulatory capacity seen in vivo. This may be true, but it is at odds with Bayesian argumentation. It may be a coincidence, but both models in which control Treg and Phd2-deficient Treg behave similarly involve treatment with anti-CD3 antibodies, raising the possibility that these antibodies in some way nullify differences reported with other stimuli, rather than this necessarily being related to the hypothesised difference between Th1 and Th17 responses in the in vivo model.
We fully agree with the reviewer’s comment, and we were similarly worried that the differences reported in vivo vs in vitro were due to different agonists used. We however attempted to evaluate Treg function in vitro using alternative approaches, including an assay in which allogeneic antigen-presenting cells (including T-cell depleted spleen cells or highly purified dendritic cells) were used as agonists and Interferon-gamma secretion and proliferation as readouts. In another set of experiments, we used in vitro or in vivo derived Th1 cells instead of naïve T cells as responders. In all instances examined to date, PHD2-deficient Tregs displayed an adequate suppressive function in vitro (data not shown).
Data showing reversal of the Phd2-deficient Treg in vivo phenotype by knockout of HIF-2alpha, but not HIF-1alpha are convincing and support the data of Yamamoto et al. The observation that Treg-specific PHD2-HIF1α double knockout mice were born at sub-mendelian ratios, displayed a marked weight loss during adult life and reduced viability, indicative of a more pronounced pro-inflammatory status is reported but data is not shown. This is certainly of interest and will no doubt receive further attention. The data that Treg-selective HIF1α or HIF2α deficiency does not affect immune homeostasis in naive mice shown in Figure S4 is relevant and compelling. These results are discussed in the context of recent work published by Hsu et al., 2020 which is interesting. Taken together these data highlight the fact that results reported throughout this manuscript arise from a combination of developmental differences with those occurring in the adult animal.
We thank the reviewer for these positive comments
The transcriptomic data presented has not, to date, been made available to reviewers or the public. Importantly, it is reported to show a disconnection between changes in glycolytic gene expression pattern and the immune phenotype. Specifically, whilst loss of Phd2 expression in Treg is associated with alterations in their regulatory function and with induction of glycolytic genes, the change in function, but not the change in glycolytic gene expression, is reversed by simultaneous knockout of HIF-2alpha and conversely the gene expression pattern, but not the change in function, is reversed by simultaneous knockout of HIF-1alpha. This will be of great interest to those working on the hypothesis that the switch between oxidative phosphorylation and glycolysis underlies functional changes in T cells, particularly if the changes in glycolytic gene expression actually convert into changes in glycolytic flux (as observed following HIF-induction in other cell types).
The transcriptomic data are available to the public on GEO with the code: GSE184581
The authors propose that a change in CXCR3 expression resulting from a change in STAT1 phosphorylation (but not absolute levels of STAT1) consequent on Phd2- inactivation leads to mal-distribution of Treg (at least in the spleen), and that given the broadly paracrine action of Treg this feature alone might explain the loss of regulatory activity in vivo. This is an intriguing hypothesis based at least in part on associative data rather than a formal proof of causality. Changes in STAT1 phosphorylation following interferon-gamma stimulation are far from 'all-or-nothing' (at the timepoint illustrated many cells have normal pSTAT1 levels even though the mean fluorescence intensity is reduced). Results in Figure 7b show that changes in STAT1 phosphorylation are seen in conventional Foxp3 negative T cells; since Phd2 knockout is restricted to the Treg lineage this change is presumably indirect, raising the possibility that the change seen in Treg is also indirect, rather than truly cell autonomous. Changes in pSTAT1 are acknowledged to affect a huge number of genes / processes so picking any one as the total explanation for any change in behaviour may be an over simplification. The analysis of changes in Treg localisation in the spleen is potentially interesting and may reach the correct conclusion but the methodology used is not clearly explained and in particular it is not clear how splenomegaly / changes in gross splenic architecture have been taken into account.
We fully agree with the reviewer comments and have now deleted the final figure of our manuscript dealing with Treg positioning in the spleen. We indeed agree that due to the morphological changes in spleen size and architecture, more detailed work would be required to confirm our initial hypothesis. Unexpectedly, and thanks to a remark from another reviewer, we found that PHD2-deficient Tregs (which are present at high frequencies in the spleen of PHD2ΔTregs mice) are largely outcompeted both in heterozygous PHD-2fl/fl Cre+/- mice (see Figure 3) and upon equal transfer into WT mice of a 1:1 mix of wt and PHD-2-deficient Tregs, greatly complicating the study of the relative positioning of these cells within lymphoid organs. We do however stand by our previous conclusion suggesting that STAT1-signaling appears as affected in PHD2-deficient Tregs. This conclusion is not only supported by the reduced accumulation of pSTAT1 in these cells, as shown in Figure 8, but also by the bioinformatic analysis of transcriptomic data and the confirmation, at the protein level, of the reduced expression CXCR3 a well characterized STAT1-dependent chemokine receptors (as shown in Figure 8).
Overall, this work contains many interesting datasets which need to be taken into account as we build our understanding of the intersection between HIF-signalling and regulatory T cell function, particularly as pharmacological manipulation of HIF signalling may provide a route to immunomodulation through alterations in regulatory T cell function.
We thank again the reviewer for this positive appreciation of our work.
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Evaluation Summary:
The possibility that local or systemic hypoxia regulates aspects of the immune system has attracted widespread interest over the last decade or more. Previous work has implicated regulatory T cells in at least some of these responses, and has demonstrated that over-activation of a specific hypoxia inducible factor (HIF) isoform, HIF-2alpha has the potential to driven pro-inflammatory lymphoproliferative responses characterized by defective regulatory T cells. The current work demonstrates that genetic activation of these hypoxia-signalling pathways that is restricted to the regulatory T cell lineage is sufficient to drive this type of immune activation. The work is important since it provides a focus for study of the mechanism, for which the authors make a proposal based on mis-localization of regulatory T cells. It …
Evaluation Summary:
The possibility that local or systemic hypoxia regulates aspects of the immune system has attracted widespread interest over the last decade or more. Previous work has implicated regulatory T cells in at least some of these responses, and has demonstrated that over-activation of a specific hypoxia inducible factor (HIF) isoform, HIF-2alpha has the potential to driven pro-inflammatory lymphoproliferative responses characterized by defective regulatory T cells. The current work demonstrates that genetic activation of these hypoxia-signalling pathways that is restricted to the regulatory T cell lineage is sufficient to drive this type of immune activation. The work is important since it provides a focus for study of the mechanism, for which the authors make a proposal based on mis-localization of regulatory T cells. It is also important in focussing a key questions (requiring further study) as to whether physiological or pathological hypoxia, specifically affecting these cells, will drive such a response and/or whether the lymphoproliferative phenotype could be affected adversely or beneficially by agents that are being used to upregulate or downregulate hypoxia-signalling pathways in other settings.
(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 #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper the authors use a conditional knockout strategy to assess the effects of deletion of the dominant oxygen-sensing hypoxia-inducible factor (HIF) hydroxylase enzyme, prolyl hydroxylase 2 (Phd2) restricted to the regulatory T cell (Treg) lineage. They use a well-established Foxp3-driven Cre recombinase allele. Phd2 is thus silenced in cells that have expressed or continue to express Foxp3 from the time this transcription factor, which is essential for Treg development and function, first occurs. They show that this approach leads to a change in Treg behaviour resulting in loss of some aspects of regulatory function and development of a Th1-like phenotype by the Foxp3 expressing cells. Effects are in general reversed when HIF-2 is silenced alongside Phd2, and may be amplified by simultaneous …
Reviewer #1 (Public Review):
In this paper the authors use a conditional knockout strategy to assess the effects of deletion of the dominant oxygen-sensing hypoxia-inducible factor (HIF) hydroxylase enzyme, prolyl hydroxylase 2 (Phd2) restricted to the regulatory T cell (Treg) lineage. They use a well-established Foxp3-driven Cre recombinase allele. Phd2 is thus silenced in cells that have expressed or continue to express Foxp3 from the time this transcription factor, which is essential for Treg development and function, first occurs. They show that this approach leads to a change in Treg behaviour resulting in loss of some aspects of regulatory function and development of a Th1-like phenotype by the Foxp3 expressing cells. Effects are in general reversed when HIF-2 is silenced alongside Phd2, and may be amplified by simultaneous silencing of the HIF-1 isoform.
The findings overlap with those reported following generalised silencing of Phd2 and following adoptive transfer of Treg in which Phd2-silencing is induced (Yamamoto et al., 2019) and are broadly compatible with those reported following a similarly Treg-restricted knockout of the von Hippel-Lindau gene (the recognition component of the E2-ubiquitin ligase that targets HIF-alpha chains that have been modified by Phd2) (Lee et al., 2015) but the results reported also differ significantly from these earlier reports in a number of intriguing respects which I feel warrant further discussion and ultimately investigation.
The Introduction is in general informative and well written but it is a shame that it does not contain more discussion of the current state of knowledge of the interplay between HIF signalling and Treg function. This would provide a platform for a more detailed and scholarly discussion of the similarities and differences between this work and existing literature in the Discussion, where existing papers are currently described rather briefly. The introduction contains the statement 'Further complexity in this pathway has been provided by the identification of additional, non-HIF-related, PHD substrates, suggesting a role of proline hydroxylation in other settings requiring oxygen-dependent regulation', citing a single reference. This does not really represent the complex balance of arguments across the literature about non-HIF substrates for the HIF hydroxylase enzymes.
The conclusions of this paper are mostly well supported by data, but some aspects need to be clarified and extended.
A central issue for any conditional knock-out strategy is whether the intended tissue restriction is successfully achieved. The authors acknowledge that some issues have been reported with the Cre-recombinase allele they use. They, however, show the expected restriction to cells of the Treg lineage in two of the lymphoid tissues under investigation (spleen and mesenteric lymph node - Supplementary figure 1b) but do not show similar results for other tissues. Some concerns arise because in Figure 8b YFP (which is expressed alongside the Cre-recombinase) is visible in what appears to be the endothelium of the spleen. Additionally, the spleen sections illustrated show convincing splenomegaly in the Phd2-deficient Treg mice but expansion of the red pulp appears to be at least as prominent as any changes that might have occurred in the white pulp. Furthermore, the gross changes in abdominal appearances described as a 'hemorrhagic abdomen' (Figure 1c) include a more plethoric abdominal wall, prominent intestinal blood vessels and a much darker, and perhaps enlarged, liver compared with the control animal. These appearances might result from increased angiogenesis and / or erythropoiesis, neither of which would be expected to result from Treg lineage restricted Phd2 knockout but are known to occur with Phd2 ablation in other tissues. If there is convincing evidence of haemorrhage it would be nice to see this more obviously displayed macro- or, perhaps better still, microscopically.
Given that the Cre-recombinase allele used is expressed through the endogenous Foxp3 locus which is located on the X-chromosome and thus subject to random inactivation in the cells of females it is important that the sex of animals used in the experiments is specified.
Experiments show alterations in Phd2-deficient Treg mice compared with control mice in homeostatic proliferation in a lymphopenic environment (Figure 3), the induction of colitis by DSS colitis (Figure 4) and the response to Toxoplasma gondii infection (Figure 4). Given the time courses these effects are likely to be real but interpretation is complicated by the spontaneous effects on the colon of Phd2-deficient Treg mice reported in Figure 1d and e. Given the wide general importance of interferon-gamma in immune / inflammatory responses I am not sure how much weight to place on the observation that concurrent interferon-gamma knockout results in loss of the Phd2-deficient Treg mice pro-inflammatory phenotype (Figure S3). No differences are seen in an in vivo model in which inflammation is induced by injection of anti-CD3 antibodies (Figure S2).
An important conceptual difference between the interpretation of results reported here and those reported by Yamamoto et al. is that the 'Phd2-deficient Treg' purified here do not show a change in regulatory function in vitro whereas those used by Yamamoto et al. failed to act normally as regulatory cells. It is unclear whether this is due to differences in the way proliferation was stimulated, the cell purification strategies used (YFP+ in the current work; CD4+;CD25+ in Yamamoto et al.), the silencing of Phd2 (by knockout throughout development here versus through an inducible-shRNA only in mature cells in Yamamoto et al.), some other feature of the experiments (e.g. the use of feeder cells) or whether a difference would be revealed by more extensive titration. The result reported here is somewhat surprising given the presence of a Th1-like immunophenotype in the cells used in these in vitro suppression assays, which at face value might mean that this immunophenotype is not responsible for changes in their regulatory capacity seen in vivo. This may be true, but it is at odds with Bayesian argumentation.
It may be a coincidence, but both models in which control Treg and Phd2-deficient Treg behave similarly involve treatment with anti-CD3 antibodies, raising the possibility that these antibodies in some way nullify differences reported with other stimuli, rather than this necessarily being related to the hypothesised difference between Th1 and Th17 responses in the in vivo model.
Data showing reversal of the Phd2-deficient Treg in vivo phenotype by knockout of HIF-2alpha, but not HIF-1alpha are convincing and support the data of Yamamoto et al. The observation that Treg-specific PHD2-HIF1α double knockout mice were born at sub-mendelian ratios, displayed a marked weight loss during adult life and reduced viability, indicative of a more pronounced pro-inflammatory status is reported but data is not shown. This is certainly of interest and will no doubt receive further attention. The data that Treg-selective HIF1α or HIF2α deficiency does not affect immune homeostasis in naive mice shown in Figure S4 is relevant and compelling. These results are discussed in the context of recent work published by Hsu et al., 2020 which is interesting. Taken together these data highlight the fact that results reported throughout this manuscript arise from a combination of developmental differences with those occurring in the adult animal.The transcriptomic data presented has not, to date, been made available to reviewers or the public. Importantly, it is reported to show a disconnection between changes in glycolytic gene expression pattern and the immune phenotype. Specifically, whilst loss of Phd2 expression in Treg is associated with alterations in their regulatory function and with induction of glycolytic genes, the change in function, but not the change in glycolytic gene expression, is reversed by simultaneous knockout of HIF-2alpha and conversely the gene expression pattern, but not the change in function, is reversed by simultaneous knockout of HIF-1alpha. This will be of great interest to those working on the hypothesis that the switch between oxidative phosphorylation and glycolysis underlies functional changes in T cells, particularly if the changes in glycolytic gene expression actually convert into changes in glycolytic flux (as observed following HIF-induction in other cell types).
The authors propose that a change in CXCR3 expression resulting from a change in STAT1 phosphorylation (but not absolute levels of STAT1) consequent on Phd2- inactivation leads to mal-distribution of Treg (at least in the spleen), and that given the broadly paracrine action of Treg this feature alone might explain the loss of regulatory activity in vivo. This is an intriguing hypothesis based at least in part on associative data rather than a formal proof of causality. Changes in STAT1 phosphorylation following interferon-gamma stimulation are far from 'all-or-nothing' (at the timepoint illustrated many cells have normal pSTAT1 levels even though the mean fluorescence intensity is reduced). Results in Figure 7b show that changes in STAT1 phosphorylation are seen in conventional Foxp3 negative T cells; since Phd2 knockout is restricted to the Treg lineage this change is presumably indirect, raising the possibility that the change seen in Treg is also indirect, rather than truly cell autonomous. Changes in pSTAT1 are acknowledged to affect a huge number of genes / processes so picking any one as the total explanation for any change in behaviour may be an over simplification. The analysis of changes in Treg localisation in the spleen is potentially interesting and may reach the correct conclusion but the methodology used is not clearly explained and in particular it is not clear how splenomegaly / changes in gross splenic architecture have been taken into account.
Overall, this work contains many interesting datasets which need to be taken into account as we build our understanding of the intersection between HIF-signalling and regulatory T cell function, particularly as pharmacological manipulation of HIF signalling may provide a route to immunomodulation through alterations in regulatory T cell function.
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Reviewer #2 (Public Review):
Despite not being the first study reporting the role of PHD2 in TREG function, Ajouaou and colleagues put together an extensive characterization of a FOXP3 conditional PHD2 KO in mice. By using 4 additional genetically modified mouse models, the authors of this study established a link between the PHD2 driven dysregulation of TREGs and another oxygen sensor, HIF2a. Both in vitro and in vivo data are compelling, and most of the hypothesis discussed in the manuscript are supported by experimental evidence (of note, the formulated hypothesis as to why the suppressive function of PHD2 KO TREGs is impaired only in vivo and appears unaffected in vitro, which might be linked with a defective co-localization between TREG and other T cells). The weakest part of this study is when authors highlight its relevance to …
Reviewer #2 (Public Review):
Despite not being the first study reporting the role of PHD2 in TREG function, Ajouaou and colleagues put together an extensive characterization of a FOXP3 conditional PHD2 KO in mice. By using 4 additional genetically modified mouse models, the authors of this study established a link between the PHD2 driven dysregulation of TREGs and another oxygen sensor, HIF2a. Both in vitro and in vivo data are compelling, and most of the hypothesis discussed in the manuscript are supported by experimental evidence (of note, the formulated hypothesis as to why the suppressive function of PHD2 KO TREGs is impaired only in vivo and appears unaffected in vitro, which might be linked with a defective co-localization between TREG and other T cells). The weakest part of this study is when authors highlight its relevance to the clinical use of oxygen sensor inhibitors, namely against PHDs, but fail to provide real evidence that these inhibitors might lead to TREG disfunction similar to that observed with the PHD2 deletion. Nevertheless, the exhaustive characterization of the PHD2-HIF2-STAT1 axis in TREGs contributes to the knowledge in the immunology field with new questions which justifies the publication of this story without further experiments.
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Reviewer #3 (Public Review):
To understand how the oxygen sensor PHD2 works in the immune system, particularly in Tregs, is of importance in understanding how the immune cells adapt to oxygen tensions in the tissue microenvironments and thereby exert their biological functions. Previous studies have shown that PHD2-HIF2a axis is critical in controlling Treg function using a systemic in vivo reversible knockdown system (Yamamoto, JCI, 2019). The current work utilized conditional PHD2 gene deletion by employing Foxp3-cre system to selectively ablate gene expression in Tregs and they found similar phenotypes like spontaneous systemic autoinflammation, activation of conventional T cells to become effector/Th1-like cells, failure to inhibit inflammatory responses in vivo using adoptive transfer or chemical/pathogen-challenged models. The …
Reviewer #3 (Public Review):
To understand how the oxygen sensor PHD2 works in the immune system, particularly in Tregs, is of importance in understanding how the immune cells adapt to oxygen tensions in the tissue microenvironments and thereby exert their biological functions. Previous studies have shown that PHD2-HIF2a axis is critical in controlling Treg function using a systemic in vivo reversible knockdown system (Yamamoto, JCI, 2019). The current work utilized conditional PHD2 gene deletion by employing Foxp3-cre system to selectively ablate gene expression in Tregs and they found similar phenotypes like spontaneous systemic autoinflammation, activation of conventional T cells to become effector/Th1-like cells, failure to inhibit inflammatory responses in vivo using adoptive transfer or chemical/pathogen-challenged models. The authors further showed that HIF2a, but not HIF1a, is the critical mediator for this function, again consistent with the previous observation. They further attempted to find the mechanisms by performing transcriptome analysis and suggested that the expression of several chemokine receptors (and others) may be responsible for the altered localization of PHD2-deficient Tregs.
Strengths (major findings):
1, in contrast to the previous systemic knockdown system, the current study employed a conditional knockout of PHD2 gene in Tregs using Foxp3-cre mice, which would give a clean system to study Tregs.
2, consistent with the previous report, the effects of PHD2 knockout in Tregs were profound, with systemic autoinflammation phenotypes. The loss of PHD2 in Tregs caused the activation of conventional CD4 T cells, which were converted into effector-Th1-like cells, the loss of in vivo suppressive activity, and the failure to control chemical-induced colitis or pathogen-driven inflammation.
3, further genetic deletion experiments showed that the effect of PHD2 is mediated by HIF2a, but not HIF1a, and the PHD2-HIF2a double deletion in Tregs reversed the above-mentioned phenotype, in general agreement with the previous publication.
4, Transcriptome analysis showed the alteration of gene expression profiles by the deletion of PHD2 alone or in different combination with HIFs, with anti-inflammatory, chemokine, or cell survival pathways as potential downstream targets. Further studies showed that Stat1 phosphorylation was affected by PHD deficiency, which was reversed by HIF2a deletion, and this may cause mis-positioning of Tregs in the spleen.
Weaknesses:
1, the major findings in this paper are largely confirmatory, with only incremental progress over the previous findings.
2, the approaches for the analysis of Tregs are not rigorous, particularly the lack of analysis of thymic Tregs, which may affect the programming of Tregs at the early stage. In addition, the extrinsic or intrinsic roles of PHD2 in Tregs in relation to the autoimmune phenotype were not strictly analyzed.
3, the claim of the reduced in vivo suppressive capacity by PHD2-deficient Tregs seems to be misleading, if the authors believe that mislocation (not really a suppression issue) is the reason for the defect. In addition, the observed Th1 phenotype in PHD2-deficient mice may not be convincingly explained by the altered location of PHD2-/- Tregs.
4, the mechanistic analyses are superficial and murky at this stage, and it does not clarify a casual vs. causal relationship between PHD2 and HIFs, or between PHD2-HIF2a axis and the downstream effectors.
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