The E3 ligase Hrd1 stabilizes Tregs by antagonizing inflammatory cytokine–induced ER stress response
Yuanming Xu, Johanna Melo-Cardenas, Yana Zhang, Isabella Gau, Juncheng Wei, Elena Montauti, Yusi Zhang, Beixue Gao, Hongjian Jin, Zhaolin Sun, Sang-Myeong Lee, Deyu Fang
Yuanming Xu, Johanna Melo-Cardenas, Yana Zhang, Isabella Gau, Juncheng Wei, Elena Montauti, Yusi Zhang, Beixue Gao, Hongjian Jin, Zhaolin Sun, Sang-Myeong Lee, Deyu Fang
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Research Article Immunology

The E3 ligase Hrd1 stabilizes Tregs by antagonizing inflammatory cytokine–induced ER stress response

Abstract

Treg differentiation, maintenance, and function are controlled by the transcription factor FoxP3, which can be destabilized under inflammatory or other pathological conditions. Tregs can be destabilized under inflammatory or other pathological conditions, but the underlying mechanisms are not fully defined. Herein, we show that inflammatory cytokines induce ER stress response, which destabilizes Tregs by suppressing FoxP3 expression, suggesting a critical role of the ER stress response in maintaining Treg stability. Indeed, genetic deletion of Hrd1, an E3 ligase critical in suppressing the ER stress response, leads to elevated expression of ER stress–responsive genes in Treg and largely diminishes Treg suppressive functions under inflammatory condition. Mice with Treg-specific ablation of Hrd1 displayed massive multiorgan lymphocyte infiltration, body weight loss, and the development of severe small intestine inflammation with aging. At the molecular level, the deletion of Hrd1 led to the activation of both the ER stress sensor IRE1α and its downstream MAPK p38. Pharmacological suppression of IRE1α kinase, but not its endoribonuclease activity, diminished the elevated p38 activation and fully rescued the stability of Hrd1-null Tregs. Taken together, our studies reveal ER stress response as a previously unappreciated mechanism underlying Treg instability and that Hrd1 is crucial for maintaining Treg stability and functions through suppressing the IRE1α-mediated ER stress response.

Authors

Yuanming Xu, Johanna Melo-Cardenas, Yana Zhang, Isabella Gau, Juncheng Wei, Elena Montauti, Yusi Zhang, Beixue Gao, Hongjian Jin, Zhaolin Sun, Sang-Myeong Lee, Deyu Fang

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Figure 6

Hrd1fl/fl-FoxP3cre mice exhibit impaired generation and function of TGF-β–converted iTregs.

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Hrd1fl/fl-FoxP3cre mice exhibit impaired generation and function of TGF-...
(A) CD4+ T cells were isolated from WT and Hrd1fl/fl-FoxP3cre mice and polarized into iTreg in vitro with 0.5, 1, or 5 ng/ml of TGF-β for 5 days. The expression of FoxP3 was analyzed by flow cytometry. (B and C) The TGF-β–converted Tregs from WT and Hrd1fl/fl-FoxP3cre mice were treated with complete medium supplemented with 5 ng/ml IL-2, with/without inflammatory cytokines IFN-γ (100 ng/ml), IL-4 (10 ng/ml), and IL-6 (50 ng/ml) for 3 days. The percentages (B) and mean fluorescent intensity (MFI) (C) of FoxP3 were measured by flow cytometry. Data are representative of 3 independent experiments. (D) Colitis progress was assessed by body weight loss (n = 6–8 per group). (E and F) Representative images of large intestine after H&E staining (E) and histological analysis (F) 10 weeks after adoptive transfer (n = 3–8 per group). Scale bars: 100 μm. (G) The total number of CD45.1+ T cells in the SPL and large intestine were measured (n = 6–10 per group). (H and I) The absolute number of IFN-γ+IL-17A–, IFN-γ+IL-17A+, and IFN-γ–IL-17A+ cells differentiated from transferred naive cells (CD45.1+) in the large intestine and SPL were calculated (n = 3–5 per group). (J) Expression level of FoxP3 from transferred Tregs (CD45.2+) in the SPL was analyzed by intracellular staining (n = 3–5 per group). (K) The absolute number of CD45.2+ cells in the SPL and large intestine were calculated (n = 3–5 per group). Data are shown as mean ± SD. *P < 0.05; **P < 0.01; and ***P < 0.005 by 2-tailed Student’s t test.