Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice

Abstract

Epstein-Barr virus-induced gene 3 (EBI3) is a widely expressed IL-12p40-related protein that associates as a heterodimer with either IL-12p35 or an IL-12p35 homologue, p28, to create a new cytokine (IL-27). To define the function of EBI3 in vivo, we generated knockout mice in which the ebi3 gene was targeted by homologous recombination. EBI3-/- mice exhibited normal numbers of both naive and mature CD4+ and CD8+ T cells and B cells, but markedly decreased numbers of invariant natural killer T cells (iNKT) as defined by staining with an alpha-galactosylceramide (alphaGalCer)-loaded CD1d-tetramer. iNKT cells from EBI3-/- mice exhibited decreased IL-4 and, to a lesser extent, IFN-gamma production after alphaGalCer stimulation in vitro. A sustained decrease in IL-4 production was also observed in EBI3-/- mice after alphaGalCer stimulation in vivo in contrast to IFN-gamma production, which was only transiently decreased under such stimulation. Notably, EBI3-/- mice were resistant to the induction of immunopathology associated with oxazolone-induced colitis, a colitis model mediated primarily by T helper (Th) 2-type cytokine production by iNKT cells. In contrast, trinitrobenzene sulfonic acid-induced colitis, a predominantly Th1-mediated colitis model, was unaffected. Thus, EBI3 plays a critical regulatory role in the induction of Th2-type immune responses and the development of Th2-mediated tissue inflammation in vivo, which may be mediated through the control of iNKT cell function.

Fig 1.

Generation of the EBI3^−/− mouse strain. (A) Exons 2–5, encoding amino acids 24–228 of the EBI3 protein, were replaced by the Neo gene cassette under control of the phosphoglycerate kinase promoter (PGK-Neo) inserted in the same orientation as the ebi3 gene. (B) Southern analysis of PvuII- and HindIII-digested genomic DNA from ganciclovir/G-418-resistant embryonic stem cell clone A12 using ³²P-labeled right (R) and left (L) probes. Clones showing the expected recombinant band at 8.4 kb with the R probe (Upper) were further screened by hybridization with the L probe (Lower), which revealed the expected recombinant band of 13.9 kb. (C) Southern analysis of genomic DNA from homozygous WT (+/+), homozygous mutant (−/−), and heterozygous (+/−) mice. BamHI-digested DNA was hybridized with ³²P-labeled EBI3 cDNA. BamHI fragments predicted to contain the left side of exon 1 (2.8 kb) are present in all three samples at equal levels, whereas fragments containing the right side of exon 1 and exons 2 and 3 (3.2 kb), and containing exons 4 and 5 (0.95 kb) are undetectable (−/− mice) or are present at half molar levels (heterozygotes). A band at ≈2.4 kb detected in all lanes corresponds to the right end of exon 5 in WT and heterozygous DNA, and to a rearranged BamHI fragment of identical size containing the right side of exon 1 linked to the Neo gene in heterozygous and knockout mouse DNA. (D) Northern blot of RNA from spleens of EBI3 WT (+/+), heterozygous (+/−), and knockout (−/−) mice hybridized to ³²P-labeled EBI3 cDNA (EBI3) or beta-actin (Actin) probes. EBI3 transcripts of 1.8 kb are detected in WT (+/+) and heterozygous (+/−) mouse RNA but are undetectable in knockout RNA (−/−).

Fig 2.

IL-4 defect in EBI3^−/− mice. (A) EBI3^−/− CD4⁺ T cells from spleen stimulated with anti-CD3 plus anti-CD28 monoclonal antibodies produce more IFN-γ in comparison with CD4⁺ T cells from WT control mice. Heterozygous mice exhibited an intermediate pattern of cytokine production. Data are representative of three experiments. (B) Similar levels of IL-12 production by DC from WT and EBI3^−/− spleens. Data are representative of two experiments. (C) CD4⁺ T cells from EBI3^−/− mice produce significantly less IL-4 than WT animals when stimulated as in A. (D) IL-4 production in EBI3^−/− mice is similar to WT mice in a Th2-inducing environment. Data are representative of three experiments. Bars represent means and standard errors of the means (n = 5). *, P < 0.05, compared with the respective WT control group. **, P < 0.002, compared with the respective WT control group.

Fig 3.

iNKT cell defect in EBI3^−/− mice. (A) Liver and spleen mononuclear cells were stained with anti-CD3 and an αGalCer-loaded CD1d tetramer. iNKT cell numbers are reduced in the liver and spleen of EBI3^−/− mice compared with WT animals. A representative flow cytometry analysis is shown. (B) Intrahepatic lymphocytes from WT (filled bars) or EBI3^−/− (open bars) were stimulated with αGalCer. IFN-γ (Right) and IL-4 (Left) levels were determined by ELISA at 24 and 48 h. EBI3^−/− iNKT cells exhibited decreased αGalCer-stimulated IL-4 production. No cytokines were detected with the control lipid βGalCer (data not shown). (C) WT (filled bars) and EBI3^−/− mice (open bars) received 2 μg of αGalCer i.p., and serum IL-4 and IFN-γ levels were assessed at 2, 4, and 24 h by ELISA. EBI3^−/− mice exhibit impaired IL-4 production after in vivo stimulation with αGalCer at all time points, whereas IFN-γ production is decreased only at 4 h after stimulation. ND, not detectable; *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

Fig 4.

Selective protection from Th2-mediated immunopathology in EBI3^−/− mice. (A) Weight loss after induction of oxazolone colitis was limited to 5% in both EBI3^−/− and EBI3^+/− mice in comparison with 15% in WT littermates. Both EBI3^−/− and EBI3^+/− exhibited complete clinical recovery on day 5 after disease induction. *, P < 0.02. (B) Almost complete absence of macroscopic signs of disease in EBI3^−/− and EBI3^+/− mice 5 days after induction of oxazolone colitis. (C) WT mice exhibit severe histopathologic evidence of colitis, whereas EBI3^−/− mice show only mild inflammatory signs. (D) Significant reduction in inflammatory score in EBI3^−/− and EBI3^+/− mice compared with WT mice. (E) Significantly reduced IL-4 production and a trend toward increased IFN-γ production of lamina propria mononuclear cells from EBI3^−/− mice in comparison with WT control mice. (F) Nuclear extracts from spleen T cells from oxazolone-treated WT (n = 3) and EBI3^−/− (n = 4) mice analyzed by Western blotting for GATA-3 (50-kDa band) expression. Control Western blotting for β-actin showed equal loading in all lanes (data not shown). (G) Similar clinical response (weight loss) after TNBS administration in EBI3^−/− and EBI3^+/− mice in comparison with WT mice. No significant weight loss was observed in an ethanol control group of WT and EBI3^−/− mice (data not shown). (H) No significant difference in pathological score among EBI3^−/−, EBI3^+/−, and WT mice in TNBS-induced colitis. Bars represent means and standard errors of the means. Data are representative of three experiments with five mice per group. *, P < 0.05 (in D and E), compared with the respective WT control groups.