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 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

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 6

Enumeration and TSDR analysis of cells bearing helper, effector, and regulatory markers in Phd2-KD and -KO mice.

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Enumeration and TSDR analysis of cells bearing helper, effector, and reg...
(A) Gating strategy and enumeration of single-positive (CD4+CD8– or CD4–CD8+), double-positive (CD4+CD8+), and double-negative (CD8–CD4–) cells within pLNs from control and shPhd2#9 mice following 4 weeks of doxycycline treatment (2 mg/mL with 30% sucrose drinking water ad libitum). **P < 0.01, by 2-tailed Student’s t test. (B) Expression of Foxp3 and CD25 within CD4+ cell populations within pLNs from control and shPhd2#9 mice following 4 weeks of doxycycline treatment. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-tailed Student’s t test. (C) CD45.2+CD3+CD4+Foxp3+ cell numbers in pLNs from lethally irradiated syngeneic CD45.1+ congenically marked mice receiving BM transplants from CD45.2+ shPhd2#9 or CD45.2+ control mice. Data are represented as the mean ± SEM. (D) CD3+CD4+Foxp3+ cell numbers in the pLNs of shPhd2#9 mice and their littermate controls, treated with doxycycline for 3 to 4 weeks and analyzed directly (ON) or 7 weeks after doxycycline withdrawal (ON/OFF) (n = 7–8 in 2 independent assays). **P < 0.01, by 2-tailed Student’s t test. (E) CD3+CD4+Foxp3+ cell numbers in pLNs from shPhd2#9 (Phd2-KD), shPhd2Hif1 (Phd2-KD/Hif1a-KD), shPhd2Hif2 (Phd2-KD/Hif2a-KD), and control mice. *P < 0.05, by 1-way ANOVA with Tukey’s multiple comparisons post hoc test. (F) CD3+CD4+Foxp3+ cell numbers in pLNs from Phd2-KO, Phd2Hif1-KO (Phd2-KO/Hif1a-KO), Phd2Hif2-KO (Phd2-KO/Hif2a-KO), and control mice. Data are represented as the mean ± SEM. (G) Flow-sorted CD4+Foxp3+ or CD4+Foxp3– cells from control or shPhd2#9 mice that were treated for 4 weeks with doxycycline were analyzed by prosequencing for the percentage demethylation of their TSDR on the active X chromosome. Data are represented as the mean ± SEM (n = at least 3/group). Unpaired, independent groups of 2 were analyzed by 2-tailed Student’s t test. Multigroup comparisons were analyzed by 1-way ANOVA with Tukey’s or Dunnett’s multiple comparisons post hoc test. TSDR data were analyzed by 2-way ANOVA with Sidak’s correction for multiple comparisons. Each symbol represents an individual mouse. Data in C–G are representative of 2 independent experiments.