Epstein-Barr virus-induced gene 3 suppresses T helper type 1, type 17 and type 2 immune responses after Trypanosoma cruzi infection and inhibits parasite replication by interfering with alternative macrophage activation

Abstract

The Epstein-Barr virus-induced gene 3 (EBI3) is a member of the interleukin-12 (IL)-12) family structurally related to the subunit p40 of IL-12 and forms a heterodimer either with the p28 subunit to build IL-27 or with p35 to form IL-35. Interleukin-27 is secreted by antigen-presenting cells whereas IL-35 appears to be produced mainly by regulatory T cells and regulatory B cells but both cytokines negatively regulate inflammatory immune responses. We here analysed the function of EBI3 during infection with the intracellular parasite Trypanosoma cruzi. Compared with C57BL/6 wild-type mice, EBI3-deficient (EBI3(-/-) ) mice showed a higher parasitaemia associated with an increased mortality rate. The EBI3(-/-) mice displayed an elevated inflammatory immune response with an increased production of T helper type 1 (Th1-), Th2- and Th17-derived cytokines. The increased Th2 immune response appears to have over-ridden the otherwise protective Th1 and Th17 immune responses by the induction of arginase-1-expressing alternatively activated macrophages in these mice. Hence, neutralization of IL-4 and arginase-1 activity partially restored protective immune responses in EBI3(-/-) mice. So far, our results demonstrate that EBI3 is an essential general regulator of inflammatory immune responses in experimental Chagas disease and is required for control of T. cruzi infection by inhibiting Th2-dependent alternative macrophage activation. Further studies are needed to dissect the underlying mechanisms and clarify whether EBI3 association with IL-27 or/and IL-35 accounts for its anti-inflammatory character in parasitic disease.

Keywords: alternative macrophage activation; interleukin-27; interleukin-35; parasitic infection.

Figure 1

Epstein–Barr virus‐induced gene 3 (EBI3) is required for control of Trypanosoma cruzi infection in vivo. (a) C57BL/6 mice were infected intraperitoneally with 500 T. cruzi blood trypomastigotes and the mRNA expression of Ebi3, Il12a and Il27 in spleens was quantified by real‐time PCR at the indicated time‐points post infection. Results are expressed as means ± SD of five mice per group and are from one of two representative experiments performed. (b) C57BL/6 and EBI3^−/− mice were infected intraperitoneally with a sublethal dose of 50 T. cruzi blood trypomastigotes. Parasitaemia and survival were assessed during the acute infection. Results are expressed as means ± SD of 10 mice per group. One out of three independent experiments with comparable results is shown. Statistical significance is shown (**P ≤ 0·01 and ***P ≤ 0·001, respectively) compared with C57BL/6 wild‐type mice.

Figure 2

Increased pathology of Epstein–Barr virus‐induced gene 3‐deficient (EBI3^−/−) mice in response to high‐dose Trypanosoma cruzi infection. C57BL/6 and EBI3^−/− mice were infected intraperitoneally with 500 T. cruzi blood trypomastigotes (a–c) or with an unphysiologically high dose of 1 × 10⁴ T. cruzi blood trypomastigotes (d–f). Organ sections of liver (a, d), heart (c) and spleen (f) were prepared at day 14 post infection and stained with haematoxylin & eosin. Representative sections from five mice per group are shown. (b, e) Liver‐derived enzymes aspartate transaminase (AST) and alanine transaminase (ALT) were quantified in sera at the indicated time‐points after infection. Data represent means ± SD of five mice per group. Similar results were obtained in two independent experiments. Statistical significance (**P ≤ 0·01) is shown compared with C57BL/6 wild‐type mice. Bar, 200 μm.

Figure 3

Trypanosoma cruzi‐infected Epstein–Barr virus‐induced gene 3‐deficient (EBI3^−/−) mice displayed increased percentages of CD4⁺ T cells, but decreased percentages of NK‐1.1⁺ T cells in spleens. C57BL/6 and EBI3^−/− mice were infected intraperiteonally with 500 T. cruzi blood trypomastigotes. (a) The total number of spleen cells was assessed over the course of infection. (b–f) Spleen cells were stained and analysed by flow cytometry at the indicated time‐points post infection. (b) The total number of CD90.2⁺ T lymphocytes in the spleens was calculated over the course of infection. The expression of T‐cell receptor‐γδ (TCR‐γδ) (c), NK‐1.1 (d) and CD4 (e) was analysed in the CD90.2⁺ T‐lymphocyte compartment. (e) The percentage of Foxp3⁺ CD25⁺ CD4⁺ spleen cells was determined. Results are expressed as means ± SD of five mice per group. One of two independent experiments with comparable results is shown. Statistical significance (*P≤ 0.05 and **P ≤ 0·01, respectively) is shown compared with C57BL/6 wild‐type mice.

Figure 4

T helper type 1 (Th1), Th17 and Th2 immune responses are simultaneously increased in Trypanosoma cruzi‐infected Epstein–Barr virus‐induced gene 3‐deficient (EBI3^−/−) mice. C57BL/6 and EBI3^−/− mice were infected intraperitoneally with 500 T. cruzi blood trypomastigotes. (a) mRNA expression of Ifng, Il17a, Il22, Il4 and Il13 in spleens was quantified by real‐time PCR at the indicated time‐points post infection. (b) Spleen cells were re‐stimulated with immobilized anti‐CD3/anti‐CD28 for 4 hr and analysed for intracellular interferon‐γ (IFN‐γ) and interleukin‐17A (IL‐17A) production at the indicated time‐points. Percentage of IFN‐γ‐ or IL‐17A‐producing cells of CD4⁺ CD44⁺ CD90.2⁺ spleen cells are shown, respectively. (c) Spleen cells were cultured for 24 hr in the presence of immobilized anti‐CD3 and IL‐17A as well as IL‐4 secretion into the supernatant was detected by cytometric bead array. Results are expressed as means ± SD of four or five mice per group and are from one of two representative experiments performed. Statistical significance (*P ≤ 0·05 and **P ≤ 0·01, respectively) is shown compared with C57BL/6 wild‐type mice. nd, not detectable.

Figure 5

Increased splenic arginase‐1 expression in Trypanosoma cruzi‐infected Epstein–Barr virus‐induced gene 3‐deficient (EBI3^−/−) mice. C57BL/6 and EBI3^−/− mice were infected intraperitoneally with 500 T. cruzi blood trypomastigotes. The mRNA expression of Nos2 (a), Arg1 and Chi3l3 (d) in spleens was quantified by real‐time PCR at the indicated time‐points post infection. (b) Serum nitrate levels were determined by Griess reaction. Results are expressed as means ± SD of four or five mice per group. Statistical significance (**P ≤ 0·01) is shown compared with C57BL/6 wild‐type mice. (c, e) Spleen sections were stained for nitric oxide synthase 2 (NOS2) (c) or arginase‐1 (e) and counterstained with haematoxylin & eosin on day 14 post infection. Representative sections from five mice per group are shown. Bar, 200 μm. Every experiment was performed two or three times, showing similar results each time.

Figure 6

Inhibition of T helper type 2 (Th2) responses reduces parasitaemia in Epstein–Barr virus‐induced gene 3‐deficient (EBI3^−/−) mice. C57BL/6 and EBI3^−/− mice were infected intraperitoneally with a sublethal dose of 50 Trypanosoma cruzi blood trypomastigotes. (a, b) EBI3^−/− mice were treated with 1 mg/ml anti‐interleukin‐4 (anti‐IL‐4) at days –1, 3, 7, 10, 14, 17 and 21 post infection. (c, d) EBI3^−/− mice were treated daily with 600 μg/200 μl N ^ω‐hydroxy‐nor‐l‐arginine (norNOHA) starting 2 days before infection. Control animals were treated with the same amount of PBS. Parasitaemia (a, c) and survival (b, d) was assessed during acute infection. Results are expressed as means ± SD of five to ten mice per group. Statistical significance (***P ≤ 0·001 and ##P ≤ 0·01, respectively) is indicated between EBI3^−/− mice and C57BL/6 mice or between EBI3^−/− and anti‐IL‐4‐treated EBI3^−/− mice, respectively.