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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

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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 1

shPhd2#9 mice develop leukocyte expansion on doxycycline treatment.

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shPhd2#9 mice develop leukocyte expansion on doxycycline treatment.
(A) ...
(A) Representative image of a shPhd2#9 mouse and its littermate control, both of which were treated with doxycycline (2 mg/mL with 30% sucrose drinking water ad libitum) for 4 to 5 weeks. (B) Representative bright-field and GFP images of pLNs from a shPhd2#9 mouse and its littermate control. Scale bars: 1 cm. (C) Wet weight of pLNs (4 per mouse) and spleens (1 per mouse) from shPhd2#9 mice (n = 6) and their littermate controls (n = 6). Each symbol in C represents an individual mouse. Data are represented as the mean ± SEM. ***P < 0.001 and ****P < 0.0001. Unpaired, independent groups of 2 were analyzed by 2-tailed Student’s t test. (D–R) Images of H&E-stained tissues from shPhd2#9 and littermate control mice: pLN, original magnification, ×2.5 (D and E), ×10 (F), and ×80 (G) (arrows in G indicate cells with oval, vesicular nuclei and eosinophilic cytoplasm); spleen, original magnification, ×5 (H and I); skin, original magnification, ×20 (J and K) and ×80 (L) (arrows in L point to exocytosis of lymphocytes into the epidermis); lung, original magnification, ×10 (M); kidney, original magnification, ×20 (N) and ×40 (O and P); liver, original magnification, ×40 (Q); and heart, original magnification, ×40 (R) (arrow in R indicates a focal collection of mononuclear inflammatory cells at the epicardial surface of the heart). Scale bars: 500 μm (original magnification, ×2.5, and proportionately shorter lengths at higher magnifications). Data are representative of 3 independent experiments at this time point.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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