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 4

Decreased IFN-γ production in EBI3-deficient CD4+ T cells attributed to reduced IL-23Rα expression.

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Decreased IFN-γ production in EBI3-deficient CD4+ T cells attributed to ...
(A–C) Naive CD4+ T cells from WT mice or EBI3-deficient mice were stimulated with plate-bound anti-CD3 and anti-CD28 under pathogenic Th17 polarization conditions for 3 days and expanded with IL-2 for more 3 days. Total cell lysates were prepared at the indicated times after stimulation and subjected to Western blotting using antibodies against EBI3, IL-23Rα, IL-12Rβ1, and actin (A). On day 5, FACS analysis for cell-surface and intracellular expression of IL-23Rα was performed (B and C). In vivo cell-surface expression of IL-23Rα and IL-12Rβ1 in CD4+ T cells of the intestinal lamina propria was also analyzed 3 weeks after transfer in the colitis model (D and E). Representative histograms and statistical difference in MFI are shown. (F) Retrovirally infected CD4+ T cells from EBI3-deficient mice with expression vectors of IL-23Rα, EBI3, or empty vector were stimulated under pathogenic Th17 polarizing conditions for 3 days, and frequency of IFN-γ+CD4+ T cells was analyzed by FACS. Data are shown as mean ± SD (n = 3, B–F) and are representative of 3 (A) or 2 (B–F) independent experiments. P values were determined using 1-way ANOVA (B–F). *P < 0.05; **P < 0.01; ***P < 0.001.