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 7

Treg functional assays.

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Treg functional assays. (A) Schematic of in vivo experimental plan. C57B...
(A) Schematic of in vivo experimental plan. C57BL/6 Rag1–/– (H-2b) mice received CD4+CD25– Teffs with or without H-2b CD4+CD25+ cells from shPhd2#9 or control mice that had received either 1 week of doxycycline (2 mg/ml in drinking water, ad libitum) pretreatment or no doxycycline pretreatment. One day later, mice received an allogeneic H-2k skin graft. Mice receiving cells from doxycycline-treated donors remained on doxycycline (B), whereas those receiving cells from untreated donors started doxycycline treatment from day 2 after transplantation (C). Allograft survival was monitored until rejection. (B) Survival graph for mice that received adoptively transferred cells derived from mice pretreated for 1 week with doxycycline. Doxycycline exposure continued in the recipient mice. (C) Survival graph for mice that received adoptively transferred cells derived from untreated mice. The cells were only exposed to doxycycline after adoptive transfer when it was added to the drinking water of recipient mice. Survival data in B and C were analyzed by log-rank test. The number of animals in each group is indicated. (D) Schematic of in vitro experimental plan. Control C57BL/6 WT Teffs (1 × 105/well) (CD4+CD25– responders [R]) were stimulated (S) with 1 × 105/well of anti-CD3 and anti-CD28 beads or 2 × 104 CBA (H-2k) DCs. CD4+CD25+ cells from shPhd2#9 or control mice were added at 1:1, 1:2, 1:4, and 1:8 ratios relative to Teff responders. Doxycycline at 1 μg/mL was added to all wells and replenished daily. Cells were incubated for 5 days, and 3H-thymidine was added for the final 18 hours of culture. Proliferation levels were normalized to the positive control (R + S). (E) In vitro suppression assay using anti-CD3 and anti-CD28 as stimulators. (F) In vitro suppression assay using DCs as stimulators. Data are represented as the mean ± SEM of biological replicates. Unpaired, independent groups (1:1, 1:2, 1:4, and 1:8) of 2 were analyzed by 2-tailed Student’s t test in E and F. *P < 0.05, **P < 0.01, and ****P < 0.0001.