Induction of suppressors of cytokine signaling by the trichothecene deoxynivalenol in the mouse

Abstract

Deoxynivalenol (DON), a trichothecene mycotoxin found in grains and cereal-based foods worldwide, impairs weight gain in experimental animals but the underlying mechanisms remain undetermined. Oral exposure to DON induces rapid and transient upregulation of proinflammatory cytokine expression in the mouse. The latter are known to induce several suppressors of cytokine signaling (SOCS), some of which impair growth hormone (GH) signaling. We hypothesized that oral exposure to DON will induce SOCS expression in the mouse. Real-time PCR and cytokine bead array revealed that oral gavage with DON rapidly (1 h) induced tumor necrosis factor-alpha and interleukin-6 mRNA and protein expression in several organs and plasma, respectively. Upregulation of mRNAs for four well-characterized SOCS (CIS [cytokine-inducible SH2 domain protein], SOCS1, SOCS2, and SOCS3) was either concurrent with (1 h) or subsequent to cytokine upregulation (2 h). Notably, DON-induced SOCS3 mRNAs in muscle, spleen and liver, with CIS1, SOCS1, and SOCS2 occurring to a lesser extent. Hepatic SOCS3 mRNA was a very sensitive indicator of DON exposure with SOCS3 protein being detectable in the liver well after the onset of cytokine decline (5 h). Furthermore, hepatic SOCS upregulation was associated with about 75% suppression of GH-inducible insulin-like growth factor acid labile subunit. Taken together, DON-induced cytokine upregulation corresponded to increased expression of several SOCS, and was associated with suppression of GH-inducible gene expression in the liver.

FIG. 1.

DON dose dependently induces SOCS mRNA expression in the spleen. Mice were treated with DON (0.1–12.5 mg/kg bw) for 2 h and spleen was analyzed for four SOCS (CIS (A), SOCS1 (B), SOCS2 (C), and SOCS3 (D)) mRNA expression by real-time PCR. Data are mean ± SEM. (n = 4–5) of mRNA fold change relative to an untreated (naïve) group (onefold). Means with asterisks differ from naïve mice (p < 0.05).

FIG. 2.

DON dose dependently induces SOCS mRNA expression in the muscle. Mice were treated with DON as in Figure 1. Real-time PCR was used to analyze gasctrocnemius muscle total mRNA for SOCS (SOCS1 (A), SOCS2 (B), and SOCS3 (C)). Data are mean ± SEM (n = 4–5) of mRNA fold change relative to an untreated (naïve) group (onefold). Means with asterisks differ from naïve mice (p < 0.05).

FIG. 3.

DON dose-dependently induces SOCS mRNA expression in the liver. Mice were treated with DON as in Figure 1. Liver was analyzed by real-time PCR for SOCS (CIS (A), SOCS2 (B), and SOCS3 (C)). Data are mean ± SEM (n = 4–5) of mRNA fold change relative to an untreated (naïve) group (onefold). Means with different letters differ (p < 0.05).

FIG. 4.

Kinetics of DON-induced cytokine and SOCS mRNA upregulation in the spleen. Mice were orally gavaged with either 12.5 mg/kg bw DON (broken lines) or PBS (solid lines). Tissues were collected 0–5 h after gavage. TNF-α (A), IL-6 (B), CIS (C), and SOCS3 mRNA (D) were analyzed by real-time PCR. Data are mean ± SEM (n = 3–4) of mRNA fold change relative to an untreated (naïve) group (onefold). Means with asterisk differ from naïve (p < 0.05).

FIG. 5.

Kinetics of DON-induced cytokine and SOCS mRNA upregulation in the liver. Mice were exposed to DON (broken line) or PBS (solid line) as in Figure 4 above. Tissues were collected 0–5 h after gavage. TNF-α (A), IL-6 (B), CIS (C), and SOCS3 mRNA (D) were analyzed by real-time PCR. Data are mean ± SEM (n = 3–4) of mRNA fold change relative to an untreated (naïve) group (onefold). Means with asterisk differ from naïve (p < 0.05).

FIG. 6.

Kinetics of DON-induced plasma proinflammatory cytokine upregulation. Mice were exposed to DON (broken line) or PBS (solid line) as in Figure 4 above. Blood was collected 0–5 h after gavage. TNF-α (A) and IL-6 (B) were analyzed by cytometric bead array. Data are mean ± SEM (n = 3–4) of plasma cytokine concentrations. Means with asterisk differ from naïve mice (0 h) (p < 0.05).

FIG. 7.

Kinetics of plasma DON concentration. Mice were exposed to DON (broken line) or PBS (solid line) as in Figure 4 above. Blood was collected 0–5 h after gavage. Plasma DON concentrations were analyzed by competitive direct ELISA. Data are mean ± SEM (n = 3–4) of DON concentrations. Means with asterisk differ from naïve mice (0 h) (p < 0.05).

FIG. 8.

Kinetics of SOCS3 protein expression in the liver. Mice were exposed to DON and PBS (vehicle), as in Figure 4 above. Histologic sections of the liver were taken at 2, 3, 4, and 5 h after DON exposure (A, B, C, and D, respectively); and 3, 5 h after vehicle exposure (E and F, respectively). Paraffin-embedded sections were immunohistochemically stained for SOCS3 and counterstained with hematoxylin after DAB reaction. Arrows indicate areas of SOCS3 protein staining. Bar = 50 μm.

FIG. 9.

Kinetics of DON's effect on hepatic IGFALS mRNA expression. Mice were exposed to DON (broken line) or PBS (solid line) as in Figure 4 above. Livers were collected at intervals (0–5 h) after gavage. IGFALS mRNAs were analyzed by real-time PCR. Data are mean ± SEM (n = 4) of mRNA fold change relative to naïve group (0 h) arbitrarily set at 100. Means with asterisk differ from vehicle at the same time (p < 0.05).

FIG. 10.

Proposed pathway for DON-induced SOCS expression and potential downstream effects. Impairment of cytokine signaling and GH-IGF1 has been demonstrated in other inflammatory models (Denson et al., 2003; Lang et al., 2005; O'Sullivan et al., 2007; Rico-Bautista et al., 2006).