EBV-induced gene 3 augments IL-23Rα protein expression through a chaperone calnexin
Izuru Mizoguchi, Mio Ohashi, Hideaki Hasegawa, Yukino Chiba, Naoko Orii, Shinya Inoue, Chiaki Kawana, Mingli Xu, Katsuko Sudo, Koji Fujita, Masahiko Kuroda, Shin-ichi Hashimoto, Kouji Matsushima, Takayuki Yoshimoto
Izuru Mizoguchi, Mio Ohashi, Hideaki Hasegawa, Yukino Chiba, Naoko Orii, Shinya Inoue, Chiaki Kawana, Mingli Xu, Katsuko Sudo, Koji Fujita, Masahiko Kuroda, Shin-ichi Hashimoto, Kouji Matsushima, Takayuki Yoshimoto
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Research Article Immunology Inflammation

EBV-induced gene 3 augments IL-23Rα protein expression through a chaperone calnexin

Abstract

Epstein-Barr virus–induced gene 3 (EBI3) is a subunit common to IL-27, IL-35, and IL-39. Here, we explore an intracellular role of EBI3 that is independent of its function in cytokines. EBI3-deficient naive CD4+ T cells had reduced IFN-γ production and failed to induce T cell–dependent colitis in mice. Similarly reduced IFN-γ production was observed in vitro in EBI3-deficient CD4+ T cells differentiated under pathogenic Th17 polarizing conditions with IL-23. This is because the induction of expression of one of the IL-23 receptor (IL-23R) subunits, IL-23Rα, but not another IL-23R subunit, IL-12Rβ1, was selectively decreased at the protein level, but not the mRNA level. EBI3 augmented IL-23Rα expression via binding to the chaperone molecule calnexin and to IL-23Rα in a peptide-dependent manner, but not a glycan-dependent manner. Indeed, EBI3 failed to augment IL-23Rα expression in the absence of endogenous calnexin. Moreover, EBI3 poorly augmented the expression of G149R, an IL-23Rα variant that protects against the development of human colitis, because binding of EBI3 to the variant was reduced. Taken together with the result that EBI3 expression is inducible in T cells, the present results suggest that EBI3 plays a critical role in augmenting IL-23Rα protein expression via calnexin under inflammatory conditions.

Authors

Izuru Mizoguchi, Mio Ohashi, Hideaki Hasegawa, Yukino Chiba, Naoko Orii, Shinya Inoue, Chiaki Kawana, Mingli Xu, Katsuko Sudo, Koji Fujita, Masahiko Kuroda, Shin-ichi Hashimoto, Kouji Matsushima, Takayuki Yoshimoto

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Figure 8

EBI3 binds to the extracellular region of IL-23Rα, but not its protective variant G149R, due to reduced binding of EBI3 to the variant.

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EBI3 binds to the extracellular region of IL-23Rα, but not its protectiv...
(A) HEK293-F cells were transfected with expression vectors of IL-23Rα-FLAG or sIL-23Rα-FLAG and EBI3 and, 48 hours later, subjected to immunoprecipitation followed by Western blotting. (B) HEK293-F cells were transfected with expression vectors of IL-12Rβ1-HA, IL-23Rα-FLAG, and EBI3, and 48 hours later, cell-surface expression of EBI3 was analyzed by flow cytometry using anti-EBI3 (solid line) or control antibody (plain line with shading). Numbers indicate percentage of EBI3+ cells. (C) HEK293T cells were transfected with expression vectors of FLAG–IL-23Rα WT or FLAG–IL-23Rα G149R and EBI3, and 48 hours later, cells were stained for cell-surface expression of IL-23Rα and then fixed and stained for intracellular expression of EBI3. Original magnification, ×600. (D and E) AD293 cells were transfected with expression vectors of FLAG–IL-23Rα-IRES-GFP WT or FLAG–IL-23Rα G149R-IRES-GFP and EBI3, and 72 hours later, cells were stained for cell-surface expression of IL-23Rα and intracellular expression of EBI3. Representative dot plots and histograms of FLAG–IL-23Rα in GFP+EBI3− and GFP+EBI3+ cells are shown (D), and the fold increase of MFI of FLAG–IL-23Rα expression was calculated (E). (F and G) HEK293T cells were transfected with expression vectors of FLAG–IL-23Rα WT or FLAG–IL-23Rα G149R and EBI3 and, 48 hours later, subjected to immunoprecipitation with anti-EBI3 followed by Western blotting with anti-FLAG (F). The intensity of each band was quantified, and the expression of IL-23Rα was normalized to EBI3 and is shown as the ratio of IL-23Rα bound to EBI3 to input IL-23Rα (G). Data are shown as mean ± SD (n = 3, D–G) and are representative of 3 independent experiments. P values were determined using 1-way ANOVA (E) or unpaired, 2-tailed Student’s t test (G). ***P < 0.005.