Cholera toxin subunit B inhibits IL-12 and IFN-{gamma} production and signaling in experimental colitis and Crohn's disease

Abstract

Background and aims: Cholera toxin B subunit (CT-B) is a powerful modulator of immune responses. The authors have previously demonstrated that oral administration of recombinant CT-B (rCT-B) is able to prevent and cure the Crohn's disease (CD)-like trinitrobenzene sulfonic acid (TNBS) mediated colitis. In this study they extended their observations and examined if rCT-B interferes with the molecular signaling underlying the Th1 type response both in TNBS colitis and in ex vivo human CD explants.

Methods: TNBS treated mice were fed with rCT-B, and IFN-gamma and IL-12 production by colonic lamina propria mononuclear cells (LPMC) was examined by ELISA. In vitro culture of mucosal explants from CD patients and non-inflammatory bowel disease controls, pre-incubated with rCT-B, were examined for IFN-gamma and IL-12 production by ELISA and semiquantitative reverse transcription polymerase chain reactions. STAT-1, -4, -6 activation and T-bet expression were examined following rCT-B treatment by western blotting both in TNBS treated mice and in human mucosal explants.

Results: rCT-B significantly reduced IL-12 and IFN-gamma secretion by LPMC from TNBS treated mice. Consistent with this, rCT-B inhibited both STAT-4 and STAT-1 activation and downregulated T-bet expression. Inhibition of Th1 signaling by CT-B associated with no change in IL-4 synthesis and expression of active STAT-6 indicating that rCT-B does not enhance Th2 cell responses. Moreover, in vitro treatment of CD mucosal explants with rCT-B resulted in reduced secretion of IL-12/IFN-gamma and inhibition of STAT-4/STAT-1 activation and T-bet expression.

Conclusions: These studies indicate that CT-B inhibits mucosal Th1 cell signaling and suggest that rCT-B may be a promising candidate for CD therapy.

Figure 1

Oral administration of rCT-B inhibits the induction of TNBS colitis and the mucosal production of IL-12 and IFN-γ. SJL mice were treated by rectal instillation with TNBS to induce colitis or with 50% ethanol alone, as colitis controls. Mice were fed with 100 μg rCT-B in 0.35 M NaHC0₃ or with 0.35 M NaHCO₃ alone as described in material and methods. (A) Determination of variations in body weight. Each point represents the cumulative mean weight data from five different experiments. In all experiments, each group consisted of at least five mice. Bars represent standard errors. *p<0.01 mice with TNBS induced colitis and concomitant administration of rCT-B/NaHCO₃ versus mice with TNBS induced colitis and concomitant administration of NaHCO₃. (B) The production of IL-12 and IFN-γ was measured in the culture supernatants of LPMC isolated from the colon of mice with TNBS colitis treated or not with oral rCT-B, or from the colon of mice treated with 50% ethanol alone. To enhance IL-12 production, LPMC were treated with SAC and IFN-γ as indicated in materials and methods. For IFN-γ production, LPMC cultures were performed in the absence of any stimulant or in the presence of anti-CD3/CD28. Data indicate mean from two independent experiments. In each experiment, cultures of pooled LPMC extracted from the colon of five mice per group were performed. Bars indicate standard error.

Figure 2

rCT-B administration inhibits IL-12 and IFN-γ signaling in mice with TNBS colitis. Total cell extracts were prepared from LPMC of mice sacrificed four days after TNBS instillation. Mice with TNBS colitis were either left untreated (TNBS) or treated with oral rCT-B (TNBS+rCT-B). As control of TNBS treatment, mice were instilled with 50% ethanol alone (Ethanol). One representative experiment of three independent experiments is shown. (A) Thirty μg of whole cell lysates were separated on a 7% SDS-PAGE, transferred to a nitrocellulose membrane and blotted sequentially with the indicated antibodies to evaluate both phosphorylation status and total content of STAT molecules. For the analysis of T-bet, 100 μg of whole cell lysates were subjected to immunoblotting with anti T-bet antibodies. After detection of T-bet, blots were stripped and incubated with a mouse anti β-actin antibody to ascertain equivalent loading of the lanes. (B) LPMC total extracts were obtained from mice with TNBS or OXA colitis either left untreated or treated with oral rCT-B (TNBS+rCT-B). Where indicated, LPMC were treated with IL-4 for 30 minutes. Thirty μg of whole cell lysates were subjected to immunoblot analysis with the indicated antibodies to evaluate both STAT-6 phosphorylation status and protein content.

Figure 3

Effect of rCT-B treatment on IL-12 expression and STAT-4 phosphorylation in ex vivo mucosal explants. Mucosal samples from inflamed (I) and uninflamed (U) tissues of a CD patient and from a non-IBD control (Ctr). T0 represents mucosal samples processed immediately as control for the basal ex vivo level and activation of the studied transcription factors. Where indicated, mucosal samples were treated for four hours with rCT-B (10 μg/ml). Thereafter, mucosal samples were washed in complete medium to remove rCT-B and incubated for additional 16–18 hours in complete medium. One representative experiment of three independent experiments is shown. (A) RNA transcripts for IL-12 p35, p40 and GaPDH were analysed in mucosal tissue omogenates by semiquantitative PCR. (B) The levels of RNA stranscripts were measured by the Fluor-S Multimager and the relative values are expressed in arbitrary units. (C) Determination of IL-12 p70 in supernatants of cultured mucosal explants. The results shown are the mean (standard error) of values obtained in three separated experiments. (D) Total proteins (500 μg/sample) were immunoprecipitated with anti-STAT-4, and immunoblotted with anti-phosphotyrosine STAT-4 (Y693). After STAT-4 p-Tyr analysis, blots were stripped and then incubated with Ab against STAT-4.

Figure 4

Effect of rCT-B treatment on IFN-γ signaling and expression in ex vivo CD mucosal samples. Mucosal samples were taken from inflamed (I) and uninflamed (U) tissues of a CD patient or from a non-IBD control (Ctr). One representative experiment of three independent experiments is shown. (A) RNA transcripts for IFN-γ and GaPDH were analysed in mucosal tissue omogenates by semiquantitative PCR. (B) The levels of RNA transcripts were measured by Fluor-S Multimager and the relative values are expressed in arbitrary units. (C) Determination of IFN-γ in supernatants of cultured mucosal explants. The results shown are the mean (standard error) of values obtained in three separated experiments. (D) Thirty μg of whole cell lysates were subjected to immunoblot analysis with the indicated antibodies to evaluate both total and phosphorylated STAT-1. T0 represents mucosal samples processed immediately as control for the basal ex vivo content of the protein. Where indicated, mucosal samples were treated for four hours with rCT-B (10 μg/ml). Thereafter, mucosal samples were washed in complete medium to remove rCT-B and incubated for additional 16–18 hours in complete medium. Total proteins (100 μg/sample) were immunoblotted with anti T-bet. After T-bet analysis, the level of β-actin was evaluated as an internal loading control.