Nrf2 Participates in M2 Polarization by Trichinella spiralis to Alleviate TNBS-Induced Colitis in Mice

Abstract

Trichinella spiralis induced alternative activated macrophages (M2), leading to protect against Crohn's disease, known as Th1 -related inflammation, which enhances oxidative stress in the host. However, the relationship of oxidative stress and T. spiralis -mediated immune response is still unknown. In our study, we showed that nuclear factor erythroid 2-related factor-2 (Nrf2), a key transcription factor in antioxidant, participated in M2 polarization induced by T. spiralis muscle larval excretory/secretory (ES) products in vitro. ES -treated M2 were injected intravenously after TNBS challenge and we demonstrated that ES-M could alleviate the severity of the colitis in mice. Adoptive transfer of ES -treated M2 decreased the level of IFN-γ and increased the levels of IL-4 and IL-10 in vivo. However, the capacity of ES -treated Nrf2 KO macrophages to treat colitis was dramatically impaired. ES -treated Nrf2 KO macrophages was insufficient to result in the elevated levels of IL-4 and IL-10. These findings indicate that Nrf2 was required for M2 polarization induced by T. spiralis ES to alleviate colitis in mice.

Keywords: Nrf2; TNBS; Th2; Trichinella spiralis; colitis; macrophage.

Figure 1

Expression of iNOS and Arg1 in wild type (WT) or Nrf2 KO macrophages induced by T. spiralis ES. Macrophages were enriched by positive selection with anti- F4/80 magnetic beads. The enriched F4/80⁺ macrophages were typically of > 90% purity as determined by flow cytometry (A). Total RNA was extracted from cells and mRNA expression levels of Arg1 (B) and iNOS (C) were quantified using RT-PCR. The results are presented as the mean ± SD of each group (n=6) *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance, as indicated by the line (one-way ANOVA with Tukey’s post hoc test).

Figure 2

Expression of CD11c (M1 marker) and CD206 (M2 marker) on wild type (WT) or Nrf2 KO macrophages induced by T. spiralis ES. The stimulated macrophages were stained PE-conjugated mAbs to CD11c (A, B) or CD206 (C, D), and analyzed by flow cytometry. Data are showed by the mean ± SD of each group (n=6) **P < 0.01, ***P < 0.001, ns, no significance, as indicated by the line (one-way ANOVA with Tukey’s post hoc test).

Figure 3

The levels of IL-12 and IL-10 secreted from wild type (WT) or Nrf2 KO macrophages induced by T. spiralis ES. Macrophages were enriched by positive selection with anti- F4/80 magnetic beads. These cells were stimulated with sterile PBS, ES or LPS alone for 24 h. And macrophages were pre-treated with ES (50 μg/ml) for 24 h before stimulation with LPS (100 ng/ml) for 24 h. Cell culture supernatants were collected and stored at −80°C. IL-12 and IL-10 levels in the supernatant were quantified by ELISA. Results are presented as the mean ± SD (n=6). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance, as indicated by line (one-way ANOVA with Tukey’s post hoc test).

Figure 4

Effect of T. spiralis ES –induced wild type (WT) or Nrf2 KO macrophages (ES-M) on TNBS-induced colitis. For adoptive transfer, WT or Nrf2 KO macrophages induced by T. spiralis ES were washed (x3) with sterile PBS, and 1 × 10⁶ cells in 500 μL of sterile PBS were injected intravenously (i.v.) after TNBS challenge. Three days later, colitis was induced by TNBS in these recipient mice. Mice were humanely euthanized by CO_2, and then the colon and spleen were collected for the following experiments. The protective efficacy against TNBS challenge was determined in three independent experiments. One representative experiment is shown here. (A) Disease activity index (DAI) was measured during the disease process. (B) The daily mean weight change in each group was calculated. (C, D) After 3 days, colons were removed, and the lengths of their colons were measured and recorded. (E) Myeloperoxidase (MPO) activity in the colonic tissues was detected. The colonic segments were stained with hematoxylin and eosin (H.E.) staining according to standard protocols. (F) The colons from each experimental group (n=6) were processed for histological evaluation (200×). (G) Histopathological damage scores were determined for the colon tissue samples. The results are representative of at least three independent experiments and expressed as the mean ± SD of each group (n=6). ***P < 0.001, ns, no significance, as indicated by line (one-way ANOVA with Tukey’s posttest) on the same day.

Figure 5

Differentiation of CD3⁺ CD4⁺ IFN-γ⁺ or IL-4⁺T cells of T. spiralis ES –induced wild type (WT) or Nrf2 KO macrophages (ES-M) on TNBS-induced colitis. (A) CD3⁺ CD4⁺ T cells were gated. IFN-γ⁺, IL-4⁺ T cells populations were determined. (B) The ratio of IL-4/IFN-γ was shown. Data are shown as the means ± SD (three independent experiments) of each group (n=6). ***p < 0.001, ns, no significance, as indicated by line (one-way ANOVA with Tukey’s posttest). These figures are representative of three independent experiments.

Figure 6

Differentiation of CD3⁺ CD4⁺ IL-10⁺T cells of T. spiralis ES –induced wild type (WT) or Nrf2 KO macrophages (ES-M) on TNBS-induced colitis. (A) CD3⁺ T cells were gated. CD4⁺IL-10⁺ T cells populations were determined. (B) The percent of CD4⁺IL-10⁺ T cells were shown. Data are shown as the means ± SD (three independent experiments) of each group (n=6). ***p < 0.001, ns, no significance, as indicated by line (one-way ANOVA with Tukey’s posttest). These figures are representative of three independent experiments.

Figure 7

Cytokine production in colons of T. spiralis ES –induced wild type (WT) or Nrf2 KO macrophages (ES-M) on TNBS-induced colitis. The colon culture supernatant was used to determine the cytokine production. IFN-γ, IL-4 and IL-10 levels were measured by ELISA. Data are shown as the means ± SD (three independent experiments) of each group (n=6). ***p < 0.001, ns, no significance, as indicated by line (one-way ANOVA with Tukey’s posttest).