EBV-induced gene 3 augments IL-23Rα protein expression through a chaperone calnexin
Izuru Mizoguchi, … , Kouji Matsushima, Takayuki Yoshimoto
Izuru Mizoguchi, … , Kouji Matsushima, Takayuki Yoshimoto
Published August 18, 2020
Citation Information: J Clin Invest. 2020;130(11):6124-6140. https://doi.org/10.1172/JCI122732.
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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 3

IFN-γ production in EBI3-deficient CD4+ T cells differentiated under pathogenic Th17 polarizing conditions with IL-23 in vitro was decreased, but not due to soluble EBI3.

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IFN-γ production in EBI3-deficient CD4+ T cells differentiated under pat...
(A and B) Naive GFP+EBI3+/+CD4+ T cells from green mice or GFP−EBI3−/−CD4+ T cells from EBI3-deficient mice (A) or an equal mixture of them (B) were stimulated with plate-bound anti-CD3 and anti-CD28 under pathogenic Th17 polarization conditions for 4 days. These cells were restimulated with PMA and ionomycin, and intracellular staining was performed. Representative dot plots of IFN-γ and IL-17 in GFP+CD4+ T cells or GFP−CD4+ T cells are shown, and the frequencies of respective CD4+ T cells were calculated. (C) Naive CD4+ T cells from WT mice or EBI3-deficient mice were stimulated with plate-coated anti-CD3 and anti-CD28 under pathogenic Th17 polarizing conditions for 4 days, and culture supernatants were collected and subjected to ELISA for detection of soluble EBI3. (D) Naive CD4+ T cells from WT mice or EBI3-deficient mice were similarly stimulated in the presence or absence of recombinant EBI3 for 4 days and subjected to intracellular cytokine staining. Data are shown as mean ± SD (n = 3–4) and are representative of 3 independent experiments. P values were determined using unpaired, 2-tailed Student’s t test (A–C) or 1-way ANOVA (D). *P < 0.05.