Inhibition of neddylation regulates dendritic cell functions via Deptor accumulation driven mTOR inactivation

Abstract

Neddylation, a newly identified post-translational modification, is significant for the activity and stability of target proteins. The exact role of neddylation in the pathogenesis of inflammatory bowel disease, specifically those mediated by dendritic cells (DCs), was still rarely reported. Here, we showed that inhibition of neddylation protected mice from mucosal inflammation. Targeting neddylation also inhibited DC maturation characterized by reduced cytokine production, down-regulated costimulatory molecules and suppressed capacity in allogeneic T cell stimulation. Additionally, inactivation of neddylation promotes caspase dependent apoptosis of DCs. These phenomena were attributed to the inactivation of mTOR, which was caused by Cullin-1 deneddylation induced Deptor accumulation. Together, our findings revealed that neddylation inhibition suppressed DC functions through mTOR signaling pathway and provided a potential therapeutic opportunity in inflammatory bowel diseases.

Keywords: Deptor; Immune response; Immunity; Immunology and Microbiology Section; dendritic cell; inflammatory bowel disease; mTOR; neddylation.

Figure 1. Neddylation inhibitor MLN4924 attenuates DSS-induced colitis in mice

A. MLN4924 prevented colon shortening in DSS-induced colitis. B. Weight loss curve for MLN4924 or cyclodextrin treated DSS mice. C. Clinical score represented colitis severity. D. Representative micrograph showed attenuated inflammation in MLN4924 treatment group compared with control group. Scale bar for 200μm. E.-F. MLN4924 reduced cytokine secretion measured by ELISA. Results were presented as the mean ± SEM. *p < .05, **p < .01, ***p < .001.

Figure 2. Inhibition of neddylation suppressed DC functions

ELISA quantification of A. TNF-α, B. IL-6 and C. IL-12p70 release in the presence or absence of vehicle, MLN4924 and LPS in BMDCs at indicated doses. D. CD80 (upper) and CD86 (lower) surface marker expression levels were declined with the risen dosage of MLN4924. One representative experiment of 3 is shown in cells treated in triplicate. *p < .05, **p < .01, ***p < .001.

Figure 3. Stimulate activity of DC is reduced in the presence of MLN4924

BALB/c BMDCs were pretreated with or without indicated dose of MLN4924. After isolation and irradiation, CD11c⁺ DCs were used as stimulators of CD4⁺ T cells and co-cultured for A. 4 days and B. 5 days at the ratio of 1:5 and 1:10. The proliferation rate of T cells was reduced with the presence of MLN4924, which means that inhibition of neddylation could decline the stimulate activity of DCs.

Figure 4. Blockade of neddylation impairs CD4⁺ T cell proliferation

CFSE-labeled CD4⁺ T cells were stimulated with anti-CD3/CD28 mAbs for A. 3 days and B. 4 days in graded dose of MLN4924 exposure, after which cell division was analyzed by flow cytometry. The results suggested that MLN4924 suppressed CD4⁺ T cell proliferation.

Figure 5. Inhibition of neddylation induces apoptosis of BMDCs

BMDCs were cultured with different doses of MLN4924 for 2 or 4 days. A. Cells were stained with Annexin V to analysis the impact of concentration and time on apoptosis. B. The curves represent the percentage of apoptosis following 2 and 4 days treatment. Trypan blue exclusion assay was employed to evaluate cell viability in 2 days C. and 4 days D. treatment.

Figure 6. MLN4924 induced apoptosis is caspase dependent

BMDCs were cultured with MLN4924. Total cellular protein extracts were prepared and subjected to immunoblotting by using A. anti-PARP, anti-caspase-3, anti-caspase-7 antibodies and B. anti-cleaved PARP, anti-cleaved-caspase-3, anti-cleaved-caspase-7 antibodies. C. Caspase-3 activity was examined by caspase activity kit. D. Immunoblotting analysis was performed for erk, p-erk, cyclin D1 and cyclin D3 expression.

Figure 7. Inhibition of neddylation triggers Deptor accumulation to inactivate mTOR signaling pathway

A. Cell lysates of the CD11c⁺ DCs sorted from the MLN4924 treated BMDCs were separated by SDS/PAGE and immunoblotted with the indicated antibodies representing mTOR signaling pathway. B. Cell lysates of the CD11c⁺ DCs were subjected to immunoblotting with anti-Deptor and anti-Cul-1 antibody (upper), and signal intensity was analyzed (lower).

Figure 8. A model for the role of MLN4924 in mTOR signaling inactivation

Neddylation inhibitor MLN4924 suppressed the conjugation of Nedd8 to Cullins and reduced SCF mediated polyubiquitination of Deptor for proteasomal degradation. The accumulation of Deptor inactivated mTOR signaling pathway, resulting in dendritic cell biological functions suppression.