Systemic silencing of Phd2 causes reversible immune regulatory dysfunction
Atsushi Yamamoto, Joanna Hester, Philip S. Macklin, Kento Kawai, Masateru Uchiyama, Daniel Biggs, Tammie Bishop, Katherine Bull, Xiaotong Cheng, Eleanor Cawthorne, Mathew L. Coleman, Tanya L. Crockford, Ben Davies, Lukas E. Dow, Rob Goldin, Kamil Kranc, Hiromi Kudo, Hannah Lawson, James McAuliffe, Kate Milward, Cheryl L. Scudamore, Elizabeth Soilleux, Fadi Issa, Peter J. Ratcliffe, Chris W. Pugh
Atsushi Yamamoto, Joanna Hester, Philip S. Macklin, Kento Kawai, Masateru Uchiyama, Daniel Biggs, Tammie Bishop, Katherine Bull, Xiaotong Cheng, Eleanor Cawthorne, Mathew L. Coleman, Tanya L. Crockford, Ben Davies, Lukas E. Dow, Rob Goldin, Kamil Kranc, Hiromi Kudo, Hannah Lawson, James McAuliffe, Kate Milward, Cheryl L. Scudamore, Elizabeth Soilleux, Fadi Issa, Peter J. Ratcliffe, Chris W. Pugh
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Research Article Immunology

Systemic silencing of Phd2 causes reversible immune regulatory dysfunction

Abstract

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes, which regulate HIFs. Genetic interventions on HIF/PHD pathways have revealed multiple phenotypes that extend the known biology of hypoxia. Recent studies have unexpectedly implicated HIF in aspects of multiple immune and inflammatory pathways. However, such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, unphysiologically restricted, and difficult to time. To study these processes better, we developed recombinant mice that expressed tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bidirectional intervention. We show that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference or inducible recombination of floxed alleles results in multilineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on reestablishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations, these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing Treg markers from these mice revealed defective function and proinflammatory effects in vivo. We believe our findings reveal a new role for the PHD2/HIF2α pathway in the reversible regulation of T cell and immune activity.

Authors

Atsushi Yamamoto, Joanna Hester, Philip S. Macklin, Kento Kawai, Masateru Uchiyama, Daniel Biggs, Tammie Bishop, Katherine Bull, Xiaotong Cheng, Eleanor Cawthorne, Mathew L. Coleman, Tanya L. Crockford, Ben Davies, Lukas E. Dow, Rob Goldin, Kamil Kranc, Hiromi Kudo, Hannah Lawson, James McAuliffe, Kate Milward, Cheryl L. Scudamore, Elizabeth Soilleux, Fadi Issa, Peter J. Ratcliffe, Chris W. Pugh

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Figure 2

Reduction of Hif2a expression reverses the phenotype in inducible Phd2-KD and Phd2-KO mice.

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Reduction of Hif2a expression reverses the phenotype in inducible Phd2-K...
(A) FACS analysis of cells from pLNs of shPhd2#9 (Phd2-KD), shPhd2Hif1 (Phd2-KD/Hif1a-KD), shPhd2Hif2 (Phd2-KD/Hif2a-KD), and control mice for CD45, CD3, B220, and Gr1 expression. Mice were treated with doxycycline (2 mg/mL with 30% sucrose drinking water ad libitum) for 4 weeks. Data from individual mice and the mean ± SEM are represented. *P < 0.05. Groups were compared by 1-way ANOVA with Tukey’s post hoc test. (B) Representative bright-field images (scale bar: 1 cm) and wet weight of pLNs from the same mice. Data are represented as the mean ± SEM. *P < 0.05 and ***P < 0.001. Multigroup comparisons were analyzed by a 1-way ANOVA with Tukey’s post-hoc test. (C) Mean BW changes (percentage relative to day 0) of the same mice during doxycycline treatment. Data are represented as the mean ± SD (n = at least 3 mice per group). Groups were compared by a repeated-measures ANOVA with Tukey’s multiple comparisons post hoc test: P < 0.001, control versus shPhd2#9; P < 0.01, control versus shPhd2Hif1; NS, control versus shPhd2Hif2; NS, shPhd2#9 versus shPhd2Hif1; P < 0.001, shPhd2#9 versus shPhd2Hif2; P < 0.001, shPhd2Hif2 versus shPhd2Hif1. (D) FACS analysis for CD45, CD3, B220, and Gr1 expression by cells in pLNs from conditional Phd2-KO, Phd2Hif1-KO (Phd2-KO/Hif1a-KO), Phd2Hif2-KO (Phd2-KO/Hif2a-KO), and control mice 4 weeks after tamoxifen treatment. Data from individual mice and the mean ± SEM are represented. Groups were compared by 1-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. (E) Representative bright-field images (scale bar: 1 cm) and wet weight of pLNs from the same mice. Data are represented as the mean ± SEM. *P < 0.05 and **P < 0.01. Multigroup comparisons were analyzed by a 1-way ANOVA with Tukey’s multiple comparisons post hoc test. Each symbol in A–E represents an individual mouse. Data were consistent over 3 independent experiments.