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 5

Transfer of the Phd2-KD phenotype by BMT.

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Transfer of the Phd2-KD phenotype by BMT. (A) Representative bright-fiel...
(A) Representative bright-field images (scale bar: 1 cm) and tissue weights of pLNs from mice that underwent BMT following 10 weeks of treatment with doxycycline (2 mg/mL with 30% sucrose drinking water ad libitum). Syngeneic congenically marked (CD45.1) mice were lethally irradiated before receiving CD45.2 shPhd2#9 BM (BMT-shPhd2#9; n = 6) or CD45.2 control BM (BMT-Ctrl; n = 2) and allowed to reconstitute for 8 weeks prior to doxycycline treatment. Data are represented as the mean ± SEM. (B) FACS analysis of cells in pLNs from BMT-Ctrl and BMT-shPhd2#9 mice, analyzed for CD45.2+, CD45.2+CD3+, CD45.2+CD19+, and CD45.2+Gr1+ expression. Data are represented as the mean ± SEM. (C) Representative fluorescence images of ANAs using mouse serum from BM transplant recipients. Scale bars: 50 μm. (D–G) H&E-stained images of (D and E) pLNs (original magnification, ×2.5) and (F and G) livers (original magnification, ×5) from BM transplant recipient mice. Arrows in F indicate inflammatory foci. Scale bars: 500 μm (original magnification, ×2.5, and proportionately shorter lengths at higher magnifications). Each symbol in A and B represents an individual mouse. The same experiment was repeated independently twice.