Systemic silencing of Phd2 causes reversible immune regulatory dysfunction
Atsushi Yamamoto, … , Peter J. Ratcliffe, Chris W. Pugh
Atsushi Yamamoto, … , Peter J. Ratcliffe, Chris W. Pugh
Published September 3, 2019; First published June 4, 2019
Citation Information: J Clin Invest. 2019;129(9):3640-3656. https://doi.org/10.1172/JCI124099.
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Research Article Immunology Therapeutics

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 9

Expression patterns of T cell–related cytokines and transcription factors in shPhd2#9 mice.

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Expression patterns of T cell–related cytokines and transcription factor...
(A) Representative flow cytometric plots showing expression of T-bet, TNF-α, IFN-γ, IL-4, GATA-3, IL-17, ROR-γt, IL-2, and IL-10 against Foxp3 within the total CD4+ cell populations from pLNs of control and shPhd2#9 mice following 4 weeks of doxycycline treatment (2 mg/ml with 30% sucrose drinking water, ad libitum). (B) Percentage of cells expressing T-bet, TNF-α, IFN-γ, IL-4, GATA-3, IL-17, ROR-γt, IL-2, and IL-10 within CD4+Foxp3– or CD4+FoxP3+ cell populations from pLNs of control and shPhd2#9 mice after 4 weeks of doxycycline treatment (n = 6 mice per group). Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-tailed Student’s t test.