A cell permeable peptide inhibitor of NFAT inhibits macrophage cytokine expression and ameliorates experimental colitis

Abstract

Nuclear factor of activated T cells (NFAT) plays a critical role in the development and function of immune and non-immune cells. Although NFAT is a central transcriptional regulator of T cell cytokines, its role in macrophage specific gene expression is less defined. Previous work from our group demonstrated that NFAT regulates Il12b gene expression in macrophages. Here, we further investigate NFAT function in murine macrophages and determined the effects of a cell permeable NFAT inhibitor peptide 11R-VIVIT on experimental colitis in mice. Treatment of bone marrow derived macrophages (BMDMs) with tacrolimus or 11R-VIVIT significantly inhibited LPS and LPS plus IFN-γ induced IL-12 p40 mRNA and protein expression. IL-12 p70 and IL-23 secretion were also decreased. NFAT nuclear translocation and binding to the IL-12 p40 promoter was reduced by NFAT inhibition. Experiments in BMDMs from IL-10 deficient (Il10(-/-)) mice demonstrate that inhibition of IL-12 expression by 11R-VIVIT was independent of IL-10 expression. To test its therapeutic potential, 11R-VIVIT was administered systemically to Il10(-/-) mice with piroxicam-induced colitis. 11R-VIVIT treated mice demonstrated significant improvement in colitis compared to mice treated with an inactive peptide. Moreover, decreased spontaneous secretion of IL-12 p40 and TNF in supernatants from colon explant cultures was demonstrated. In summary, NFAT, widely recognized for its role in T cell biology, also regulates important innate inflammatory pathways in macrophages. Selective blocking of NFAT via a cell permeable inhibitory peptide is a promising therapeutic strategy for the treatment of inflammatory bowel diseases.

Figure 1. Tacrolimus inhibits IL-12 p40 protein and mRNA expression.

(A) Murine bone marrow-derived macrophages (BMDMs) were pretreated with the indicated concentrations of tacrolimus for 1 h. Cells were then either left untreated (NS) or stimulated with LPS (100 ng/ml) or LPS and IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (B) BMDMs were incubated with the indicated concentrations of tacrolimus for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml, where indicated) prior to LPS (100 ng/ml) stimulation for 4 h or left untreated (NS). Cells were harvested and total RNA was assayed for Il12b mRNA levels by real-time RT-PCR. Each result represents the mean ± standard error (SD) for duplicate assays from three independent experiments. * p<0.05.

Figure 2. 11R-VIVIT inhibits IL-12 p40 protein and mRNA expression.

(A) Murine bone marrow-derived macrophages (BMDMs) were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide 11R-VEET for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (B) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for Il12b mRNA levels by real-time RT-PCR. Results were normalized with the respect to the levels of β-actin mRNA, and represent the mean ± SE for duplicate assays from three independent experiments. * p<0.05, ** p<0.01.

Figure 3. 11R-VIVIT inhibits pro-inflammatory cytokine expression in murine macrophages.

BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 (A), IL-23 (B) and TNF (C) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p<0.05.

Figure 4. 11R-VIVIT reduces DNA binding to the NFAT/IRF8 site in the murine IL-12 p40 promoter.

BMDMs were either untreated (lane 1) or pretreated with the indicated concentrations of 11R-VEET (lanes 2–4) or 11R-VIVIT (lanes 5–7) for 1 h. Cells were then treated for 1 h with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) stimulation for 4 h. Cells were harvested and nuclear extracts were prepared for EMSA. ³²P labeled oligonucleotide probe spanning the NFAT/IRF8 site of the IL-12 p40 promoter was incubated with 10 mg of nuclear extracts on ice for 30 min prior to electrophoresis. For supershift assays (lanes 3,4 and 6,7), 10 mg nuclear extract were incubated with 3 mg of anti-NFAT c1 (lanes 3 and 6) or anti-IRF8 (lanes 4 and 7) antibodies for 30 min on ice prior to addition of the ³²P labeled probe and 30 min incubation on ice followed by electrophoresis. The arrows represent DNA-protein complexes formed before and after incubation with the indicated antibodies. The above result is representative of five independent experiments.

Figure 5. NFAT binds to Nos2 promoter and its selective inhibition abrogates nitric oxide secretion in macrophages.

(A) Nuclear extracts were prepared from BMDMs stimulated with LPS (lane 1) or LPS and IFN-γ (lanes 2–7). Extracts were either incubated with a labeled probe NFAT/ISRE (element from region II of the promoter scheme), a competitor ISRE oligonucleotide, or with the indicated antibodies. DNA-protein complexes (indicated by arrow) were separated by electrophoresis. EMSA revealed binding of both IRF8 and NFATc1 to the same Nos2 promoter element. (B) BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitric oxide secretion was assayed from supernatants by Greiss reaction. Experiments were performed in duplicate and repeated three times (mean ± SEM). (C) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with LPS alone or IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for Nos2 mRNA levels by real-time RT PCR. Data is representative of three independent experiments. * p<0.05.

Figure 6. Inhibition of IL-12 p40 by 11R-VIVIT is independent of IL-10.

(A) Murine BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT (white bars) or 11R-VEET (black bars) for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-10 protein secretion was assayed from supernatants by ELISA. (B) BMDMs from Il10 ^−/− mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and LPS plus IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. Results represent the mean ± SD for duplicate assays from three independent experiments. * p<0.05, ** p<0.01.

Figure 7. Therapeutic effect of VIVIT on piroxicam induced colitis in Il10 ^−/− mice.

Piroxicam fed IL-10 deficient mice were injected with the indicated doses of either VIVIT or VEET peptides (6–7 mice per group) every other day for two weeks. Colons were then harvested and colitis scores were determined as described in materials and methods. (A–B) An inverse dose response of VIVIT therapy was noted, with a higher mortality rate at 10 mg/kg (A) and amelioration of colitis detected at as low as 0.5 mg/kg following one week of piroxicam diet (B). (C) 1 mg/kg of either peptide was then administered to a group of 21 piroxicam fed Il10 ^−/− mice. The scores for histological damage during piroxicam-induced colitis were significantly higher in VEET treated mice compared to those receiving VIVIT peptide in all reported experiments. Data shown are mean ± SEM.

Figure 8. VIVIT treatment reduces spontaneous secretion of colonic inflammatory cytokines.

Explants from colons of VIVIT (n = 12 for A, B; n = 7 for C) and VEET (n = 9 for A, B; n = 6 for C) treated mice were incubated in RPMI medium for 24 h, released cytokines were then determined by ELISA. We noted a marked difference in the gut spontaneous secretion of TNF-α (B) in gut supernatants from VIVIT treated mice, with a statistically significant change in IL-12 p40 (A) and in interferon-γ (C) production compared to VEET treated mice. Values are normalized to weight of intestinal explants, and data are presented as mean ± SEM.