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 7

EBI3 binds to calnexin and IL-23Rα, and calnexin is necessary for the EBI3-mediated augmentation of IL-23Rα protein expression.

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EBI3 binds to calnexin and IL-23Rα, and calnexin is necessary for the EB...
(A) HEK293T cells were transfected with expression vectors of IL-23Rα-FLAG and EBI3, and 48 hours later, total cell lysate was prepared and subjected to immunoprecipitation with anti-FLAG or anti-EBI3, followed by Western blotting with anti-calnexin, anti-EBI3, and anti-FLAG, as indicated. (B and C) HEK293T cells were transfected with expression vectors of IL-23Rα-FLAG, EBI3, and mouse calnexin-MYC, and 72 hours later, cells were fixed for intracellular staining of IL-23Rα using anti–IL-23Rα followed by secondary antibody conjugated with Alexa Fluor 488, for EBI3 using anti-EBI3 followed by secondary antibody conjugated with Alexa Fluor 594, and for calnexin using anti-calnexin followed by secondary antibody conjugated with Alexa Fluor 647 (B). For cell-surface staining, the fixation procedure was omitted (C). Representative photographs are shown. Original magnification, ×600. (D and E) WT and calnexin KO HEK293T cells were transfected with expression vectors of IL-23Rα-FLAG, IL-12Rβ1-HA, and EBI3. After 72 hours, total cell lysate was prepared for Western blotting. (D), intensity of each band of IL-23Rα-FLAG was detected by anti-FLAG or anti–IL-23Rα, and actin was quantified, and the expression of IL-23Rα-FLAG was normalized to actin and is shown as the relative expression to respective untransfection (E). (F) Immunoprecipitation analyses with anti-EBI3 followed by Western blotting with anti–IL-23Rα or anti-calnexin were performed using cell lysates of WT naive CD4+ T cells stimulated under pathogenic Th17 polarizing conditions for 3 days. Note each blot was run in parallel and contemporaneously. Data are shown as mean ± SD (n = 3, D and E) and are representative of 2 (D–F) or 3 (A–C) independent experiments. P values were determined using unpaired, 2-tailed Student’s t test (E). *P < 0.05; **P < 0.01; ***P < 0.005.