Vasoactive intestinal peptide induces regulatory dendritic cells with therapeutic effects on autoimmune disorders

Abstract

The induction of antigen-specific tolerance is critical for the prevention of autoimmunity and maintenance of immune tolerance. In addition to their classical role as sentinels of the immune response-inducing T cell reactivity, dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Tr). The possibility to generate tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Therefore, the characterization of the endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. In this study, we report on the use of the known immunosuppressive neuropeptide, the vasoactive intestinal peptide, as a new approach to induce tolerogenic DCs with capacity to generate Tr cells, to restore tolerance in vivo, and to reduce the progression of rheumatoid arthritis and experimental autoimmune encephalomyelitis.

Fig. 1.

VIP induces a stable “semimature” phenotype in BM-DCs. DCs were generated from mouse BM cells in the absence (DC_control) or presence (DC_VIP) of VIP and activated with LPS to induce DC maturation. (a)DC_control and DC_VIP were double-labeled for different markers and analyzed by flow cytometry. Numbers represent the percentage of positive cells (n = 4). (b) Cytokine content in the DC supernatants was determined by ELISA (n = 4).

Fig. 2.

Murine DCs differentiated in the presence of VIP induce regulatory Tr1 cells and tolerance in vivo. DCs were generated from mouse BM cells in the absence (DC_control) or presence (DC_VIP) of VIP and activated with LPS to induce DC maturation. (a)DC_control or DC_VIP was added to allogeneic CD4 T cells (5 × 10⁵), and the proliferative response was determined. DC_control or DC_VIP without T cells did not proliferate. Each result is the mean ± SD of three experiments performed in duplicate. (b) Purified CD4 T cells were exposed to allogeneic DC_control or DC_VIP and activated with phorbol 12-myristate 13-acetate plus ionomycin. Intracellular cytokines were determined in CD4-gated cells by flow cytometry. Numbers represent percentage of positive cells (n = 5). (c) Purified CD4 T cells were stimulated for 1 week with allogeneic DC_control or DC_VIP. The resulting regulatory CD4 T cells (Tr) were incubated with syngeneic responder CD4 T cells (rCD4) in the presence of allogeneic mDCs, and the proliferative response was determined (n = 4). (d) Isolated CD4 T cells were cocultured with syngeneic Tr_VIP and allogeneic mDCs in the presence or absence of blocking anti-IL10 and/or anti-TGFβ. Additionally, CD4+mDCs were separated from Tr_VIP+mDC in a Transwell system. The proliferative response of responder CD4 T cells was determined (n = 4). (e) Sorted CD4 T cells generated with DC_control or DC_VIP were analyzed for neuropilin 1 and Foxp3 mRNA expression by real-time RT-PCR and for surface CD103 and glucocorticoid-induced TNF receptor (GITR) expression by flow cytometry. Open histograms and dashed lines represent isotype controls. One representative experiment of two is shown. (f) Mice were injected s.c. with increasing numbers (from 50 to 5 × 10⁵ cells) of Ag-pulsed DC_control or DC_VIP 1 week before priming with Ag. Five days later, mice were tested for DLN Ag-specific T cell proliferation, serum antibody levels, and DTH responses. Mice injected with Ag alone (None) were used as controls. Results are the mean ± SD for each group (n = 4) tested separately and are representative of three experiments.

Fig. 3.

Therapeutic effect of DC differentiated with VIP in RA and EAE. (a) DBA1/J mice (H-2^q) with established CIA or C57BL/6 mice (H-2^b) with established EAE were treated (arrows) with syngeneic CII-pulsed DCs or MOG-pulsed DCs, respectively, generated in the absence (DC_control, ○) or presence (DC_VIP, ▾ of VIP. Untreated CIA and EAE mice (none, •) were used as controls. Clinical score was monitored (n = 12). (b) CII- and MOG-pulsed DC_VIP were injected at different doses. (c) CII-induced proliferation and IFNγ production by spleen T cells, and the levels of anti-CII IgG in sera were determined in CIA mice injected with DC_control or DC_VIP (n = 5). (d) The effect of DC_VIP is Ag-specific. Arthritic mice were treated with unpulsed, CII-pulsed, or OVA-pulsed DC_control or DC_VIP after disease onset. One week later, mice were immunized s.c. with OVA or CII and challenged i.d. in the ear pinna with the respective Ag 5 d later. Clinical score and DTH responses were determined 24 h later (n = 5). (e) Untreated CIA or EAE mice or animals injected with DC_VIP and treated with control Ig, anti-IL10, anti-TGFβ, or a combination of both mAbs (10 mice per group).

Fig. 4.

DC_VIP-induced Tr prevent autoimmunity. (a) Therapeutic effect on arthritis. CIA (H-2^q) or EAE mice (H-2^b) with established disease were treated with syngeneic CII- and MOG-specific Tr_control (○) or with different doses of CII- or MOG-specific Tr_VIP (10⁶ cells, ▾ ;5 × 10⁵ cells, ▿;5 × 10⁴ cells, ▪). Untreated mice (•) were used as CIA and EAE controls. Clinical score was determined (n=10). (b) Untreated CIA/EAE mice or CIA/EAE mice injected with Tr_VIP and treated with control Ig, anti-IL10, anti-TGFβ, or anti-IL10 plus anti-TGFβ Abs. Clinical score was measured at the peak of the disease (n = 10).