Brain mononuclear cells
Draining lymph node
Regulatory T cells
Vasoactive intestinal peptide
CD4+CD25+ regulatory T cells (Treg) control the immune response to a variety of antigens, including self-antigens. Several models support the idea of the peripheral generation of CD4+CD25+ Treg from CD4+CD25– T cells. Little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+CD25+ Treg. In this study we report on the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, to induce functional Treg in vivo during the development of experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. The administration of VIP to EAE mice results in the expansion of the CD4+CD25+, Foxp3-expressing T cells in the periphery and the nervous system, which inhibit encephalitogenic T cell activation. In addition to the increase in the number of CD4+CD25+ Treg, VIP induces more efficient suppressors on a per cell basis. The VIP-generated CD4+CD25+ Treg transfer suppression and significantly ameliorate the progression of the disease.
The immune system is faced with the daunting job of protecting the host from an array of pathogens, while maintaining tolerance to self-antigens. The induction of antigen-specific tolerance is essential to maintain immune homeostasis, to control autoreactive T cells and preventing the onset of autoimmune diseases. Thymic selection prevents to a large degree the release of functional autoreactive T cells. However, potential autoreactive T cells persist in the periphery of healthy individuals and retain the capacity to initiate autoimmune disease. Thus, peripheral regulatory mechanisms are required to protect against self-directed immune responses. Active suppression by CD4+CD25+ regulatory T cells (Treg) plays a key role in the control of self-reactive T cells, and in the induction of peripheral tolerance 1, 2.
Two major types of Treg cells have been characterized in the CD4+ population, i.e. the naturally occurring, thymus-generated Treg, and the peripherally induced, IL-10 or TGF-β secreting Treg 1–3. The CD4+CD25+, Foxp3-expressing, naturally occurring Treg generated in thymus, migrate and are maintained in the periphery. The signals for their thymic generation and maintenance in the periphery are not entirely defined. The number of CD4+CD25+ Treg in the periphery does not decrease with age, although these cells are anergic and prone to apoptosis, and their site of origin, the thymus, undergoes age-related involution. This suggests that CD4+CD25+ Treg can be generated peripherally. Indeed, several experimental models support the idea of peripheral generation of CD4+CD25+ Treg from CD4+CD25– T cells 4. The endogenous factors and mechanisms controlling the peripheral expansion of CD4+CD25+ Treg are mostly unknown.
Vasoactive intestinal peptide (VIP) is a potent Th2-produced immunosuppressive agent that proved to be protective in several models of autoimmune diseases such as collagen-induced arthritis, inflammatory bowel disease, uveoretinitis 5–7, and EAE (submitted for publication). Until now, the mechanisms described for the VIP immunosuppressive activity included the deactivation of macrophages, DC, and microglia, and promoting Th2 effector differentiation and survival 8. In the present work, we investigated whether VIP treatment might exert its EAE-protective effect by increasing the generation/activation of the Treg compartment.
VIP treatment induces the emergence of Treg CD4+CD25+ cells during EAE
We have recently shown the therapeutic effect VIP on EAE (submitted for publication). This effect is associated with a striking reduction of the two deleterious components of the disease, i.e. the autoimmune and inflammatory response. VIP decreases the presence of encephalitogenic Th1 cells in the periphery and the CNS. In addition, VIP strongly reduced the inflammatory response during EAE progression by down-regulating the production of several inflammatory mediators in both spinal cord and brain parenchyma. Several studies have indicated that Treg cells confer significant protection against EAE, by promoting protective Th2 responses and decreasing the CNS homing of autoreactive cells 9–12. Because the VIP treatment also inhibited events in the inflammatory phase of EAE following the activation of antigen-specific CD4 Th1 cells, there is a possibility that VIP induces Treg with suppressive activity during the progression of the disease. In this sense, CD4 T cells from VIP-treated EAE mice did not transfer the disease (Fig. 1A). However, when these cells were depleted of CD4+CD25+ cells prior to transfer, they were able to transfer the disease (Fig. 1B). This suggests that VIP might induce the generation and/or activation of Treg. Therefore, we investigated whether VIP induces Treg during EAE. VIP-treated EAE mice had higher percentages of CD4+CD25+ cells in both draining lymph nodes (DLN) and brain compared to control EAE mice (Fig. 2A). VIP-induced CD4+CD25+ cells exhibit an activated Treg phenotype 13–15, i.e. CD45RBlowCD62LhighCD69highCTLA-4high (Fig. 2A). CD4+CD25+ cells isolated from VIP-treated mice also express higher levels of CTLA-4, a key player in Treg function (Fig. 2A). Although Treg constitutively express CD25 and CTLA-4, these receptors are also expressed on activated effector T cells. Several other markers have been recently identified in Treg, e.g. Neuropilin-1 (Nrp1), the transcription factor Foxp3, and the glucocorticoid-induced TNF receptor (GITR)13–16. We found that LN CD4 cells isolated from VIP-treated EAE mice express higher levels of Foxp3, Nrp1 and GITR (Fig. 2B) than those isolated from control EAE mice. In addition, whereas control EAE mice did not show any Foxp3+ cells in the CNS areas extensively infiltrated by CD4 T cells, spinal cords of VIP-treated mice showed increased numbers of Foxp3+ cells, mainly localized in the perivascular cuffs (Fig. 2C). The changes in Foxp3, Nrp1 and GITR expression induced by VIP in the CD4 population were uniquely due to an increase in the number of CD4+CD25+ Treg cells (20.2% for VIP versus 5.1% for control), but not to changes in levels of expression per cell, because LN CD4+CD25+ isolated from untreated and VIP-treated EAE mice expressed the same amounts of Foxp3, Nrp1 and GITR (not shown). These data suggest that VIP treatment promotes the generation of activated Treg during EAE.
To test the specificity of the VIP effect and to identify the VIP receptor involved in this effect, we used specific VIP agonists. The VPAC1 agonist mimicked the VIP effects increasing the numbers of CD4+CD25+ Treg on EAE (12.4 in LN and 1.7% in CNS), whereas the VPAC2 agonist showed only a weak effect (3.1 in LN and 0.2% in CNS). This is in agreement with the respective therapeutic effect of both VIP agonists on EAE (submitted for publication).
When stimulated, Treg suppress the proliferation and IL-2 production of antigen-specific effector T cells. Several mechanisms have been identified for Treg function, such as surface CTLA-4 and TGF-β expression, costimulatory blockade, and release of IL-10 and/or TGF- β 1–4. Naturally occurring CD4+CD25+ Treg exert their suppressive activity primarily through direct cellular contact, whereas peripheral T suppressors act primarily through cytokines1–3. To determine whether CD4 T cells isolated from VIP-treated EAE mice function as suppressive Treg, we co-cultured CD4 cells from EAE mice treated with medium (CD4control) or VIP (CD4VIP) with CD4 cells from EAE mice (responder rCD4) in the presence of antigen presenting cells and antigen proteolipid protein (PLP). CD4control did not suppress the proliferation of rCD4 cells, and slightly up-regulated IL-2 and IFN-γ production in response to antigen (Fig. 3A). In contrast, CD4VIP suppressed the proliferation of autoreactive rCD4 (Fig. 3A). The suppression increased with the number of CD4VIP, being effective even at a ratio as low as one CD4VIP to eight rCD4 cells (Fig. 3B). CD4VIP also inhibited IL-2 and IFN-γ production, while increasing the levels of the regulatory cytokines IL-10 and TGF- β (Fig. 3A). We addressed the question whether CD4VIP inhibit encephalitogenic T cells through direct cellular contact and/or soluble factors. When CD4VIP and autoreactive CD4 cells were separated in transwell experiments, the suppressive activity was partially abolished, indicating that both direct contact and soluble factors mediate the inhibitory effect (Fig. 3C). In regular cocultures, addition of anti-TGF- β , anti-IL-10, or anti-CTLA-4 Ab reversed inhibition modestly. However, blocking both IL-10 and TGF- β had a more pronounced effect, and addition of all three Ab (anti-IL-10, anti-TGF- β , and anti-CTLA-4) reversed the inhibitory effect completely (Fig. 3C). In addition, as previously described for Treg, exogenous IL-2 overcame the suppressive activity (Fig. 3C). These results demonstrate that VIP administration during EAE induces the generation and/or activation of Treg cells that efficiently suppress autoreactive CD4 T cells.
VIP induces the generation of peripheral CD4+CD25+ Treg from CD4+CD25– T cells
CD4+CD25+ Treg can be generated peripherally from CD4+CD25– T cells 4. To determine whether the VIP-induced increase in CD4+CD25+ Treg during EAE is due to the expansion of the existing naturally occurring CD4+CD25+ Treg, or to newly generated Treg from CD4+CD25– T cells, we depleted the EAE mice of CD4+CD25+ T cells by treatment with anti-CD25 Ab before VIP inoculation. Depletion of CD25+ T cells prior to EAE induction resulted in a more severe disease than in controls, with an earlier onset and higher clinical scores (five out of six animals died by day 9, and the last mouse died on day 20, whereas all six mice in the control group survived) (Fig. 4A). In contrast, CD25+ depletion did not affect the beneficial effect of VIP (Fig. 4A). At the time of maximum clinical score in the control group (day 15), the EAE mice depleted of CD25+ cells and treated with VIP possessed almost the same number of splenic CD4+CD25+Foxp3+ T cells as the VIP-treated un-depleted mice (10 and 12%, respectively) (Fig. 4A, right panel). This is in contrast to the EAE controls not treated with VIP (2% CD4+CD25+Foxp3+ cells). These experiments suggest that VIP induces the generation of peripheral CD4+CD25+ Treg from CD4+CD25– T cells. To further confirm this hypothesis, CD4+CD25– cells isolated from DLN of EAE mice at the peak of the disease were stimulated invitro with anti-CD3/CD28 in the absence or presence of VIP. The incubation with VIP significantly increased the percentage of CD4+CD25+ cells and the levels of Foxp3 in the cultures (Fig. 4B), suggesting that VIP could induce the peripheral generation of CD4+CD25+ Foxp3+ cells from the CD4+CD25– compartment.
Involvement of Treg in the therapeutic effect of VIP in EAE
We next tested in vivo the function of VIP-generated Treg in the adoptive transfer EAE model, by administering CD4control and CD4VIP T cells together with activated PLP-specific, CFSE-labeled rCD4 to naive recipients. In contrast to CD4control, CD4VIP T cells prevented the adoptive EAE transfer (Fig. 5A and B). The CD4VIP-induced clinical improvement correlates with the reduction in the number and the proliferation of encephalitogenic CD4 cells (CFSE-labeled) in both spleen and CNS (Fig. 5C). This suggests that the VIP-induced Treg suppressed the activation and proliferation of autoreactive CD4 cells in the periphery. Removal of CD4+ or CD4+CD25+ T cells from the CD4VIP abrogated or significantly reduced the protective action, whereas co-transfer of isolated CD4+CD25 prevented EAE in the recipients (Fig. 5D). The therapeutic effect of CD4VIP and CD4+CD25 was associated with the down-regulation of the autoimmune component of the disease, because DLN T cells from CD4- and CD4+CD25-treated mice showed weak proliferation and IFN-γ production in response to the autoantigen (Fig. 5E).
EAE is an inflammatory, autoimmune demyelinating disease of the CNS, which shows pathologic and clinical similarities to human multiple sclerosis (MS) and is used as a model to test potential therapeutic agents17. Both EAE and MS are considered archetypal CD4 Th1 cell-mediated autoimmune diseases in which Th1 cells reactive to components of the myelin sheath, infiltrate the CNS parenchyma, release proinflammatory cytokines and chemokines, and promote macrophage infiltration and activation17. Inflammatory mediators such as cytokines and free radicals, produced by infiltrating cells and resident microglia, play a critical role in demyelination, contributing to oligodendrocyte loss and degenerative axonal pathology18. Although available therapies based on immunosuppressive agents inhibit the inflammatory component of MS and either reduce the relapse rate or delay disease onset, they do not suppress progressive clinical disability. The autoimmune-protective action of VIP have been demonstrated in a variety of contexts, by reducing pathological Th1 responses and deactivating microglia, DC and macrophages 5–8. Our results demonstrate that VIP induces the generation and/or activation of efficient CD4+CD25+ Treg during EAE. CD4+CD25+ Treg have been reported to play a critical role in the regulation of autoimmune diseases, including MS 1, 2, 9–12. Of physiological relevance is the fact that VIP administration to EAE mice induced the appearance of CD4+CD25+ cells with a Treg phenotype in the CNS (Fig. 2A). The Treg induced by VIP in the periphery apparently could cross the blood-brain-barrier, accumulate in the CNS, and induce suppression of the encephalitogenic response.
CD4+CD25+ Treg have been characterized by high expression of the transcription repressor Foxp3, high surface expression of GITR, Nrp1, CD103, CD62L and CD69, and low expression of CD45RB 13–16. The CD4 population from VIP treated EAE mice showed a decrease in CD45RB, and increases in the expression of all the other markers, compared to the CD4 T cells from antigen-inoculated mice. In addition to expanding the CD4+CD25+ population, VIP also induced more efficient Treg, both in terms of cytokine secretion and suppressive activity. The VIP-induced CD4+ Treg cells produce high levels of IL-10 and TGF- β . In addition, on a per cell basis, the VIP-induced CD4+ Treg are very strong suppressors of responder autoreactive T cell proliferation, particularly at low regulatory T cell/autoreactive T cell ratios. Although the VIP-induced CD4+CD25+ Treg secrete IL-10 and TGF- β , they also inhibit autoreactive T cell proliferation through direct cellular contact. This distinguishes the VIP-induced Treg from the classical Tr1/Th3(Tr2) regulatory CD4+ T cells, whose suppressive mechanism is cytokine-dependent 19–21, and from the recently reported CD25+ cell-cell contact-dependent and cytokine-independent suppressors recruited from the peripheral CD25– population by CD4+CD25+ T cells stimulated with IL-2 and TGF- β 22, suggesting that the Treg population induced by VIP during EAE could be a novel Treg population that resemble to different populations of Treg cells. Alternatively, VIP could induce/activate the different types of Treg already described, which cooperatively act in the suppressive response. In this sense, we have recently described that VIP induce the differentiation of tolerogenic DC with capacity to generate Tr1-like cells with regulatory function in vitro and in vivo23, 24. In addition, the administration of tolerogenic DC differentiated by VIP in vitro inhibited the progression of EAE by inducing Tr1-like cells in the host 24. The mechanisms involved in the generation/activation of Treg by VIP during EAE are not fully understood. However, the present study shows evidence that they can be peripherally generated from CD4+CD25– cells, because VIP treatment prevented EAE progression in CD25-depleted mice and restored the number of CD4+CD25+ Treg cells, and VIP induces the in vitro generation of CD4+CD25+Foxp3+ cells from activated CD4+CD25– EAE cells. Therefore, our hypothesis is that VIP is able to directly generate CD4+CD25+Foxp3+CTLA-4+ Treg cells from the peripheral CD4+CD25– compartment, and to indirectly generate IL-10/TGFβ-secreting Tr1-like cells through the induction of tolerogenic DC 23, 24, and the cooperation between both Treg cells would contribute to the therapeutic effect of VIP on autoimmune disorders.
A characteristic marker of Treg cells is the constitutive expression of CTLA-4, a negative regulatory factor critical for the induction and function of Tr cells 25. In agreement with these reports, VIP-induced Treg express high levels of CTLA-4, explaining the partial dependence of cell-cell contact in the regulatory activity of these cells. In addition, CTLA-4 plays a role in the suppressive mechanism of VIP-induced CD4+CD25+ Treg, since suppression is abrogated by treatment with anti-CTLA-4 Abs. Although CTLA-4 is expressed at high levels in Treg, its role in the development and/or function of Treg is not clear. A mechanism involving induction of indoleamine 2,3-dioxygenase following CTLA-4 binding to B7 has been recently proposed 26.
Recently considerable effort has been focused on the use of antigen-specific Treg generated exvivo for the treatment of several autoimmune diseases. We have found that treatment with VIP-induced Treg suppresses encephalitogenic T cells and prevents the progression of the disease. The generation of highly efficient Treg by VIP ex vivo could be used as an attractive therapeutic tool in the future, avoiding the administration of the peptide to the patient. Interestingly, VIP mimics some of the suppressive effects of glatiramer acetate (Copaxone) in EAE, a synthetic polypeptide drug approved for the treatment of MS, whose therapeutic actions involve anti-inflammatory effects and Treg induction27. These observations provide a powerful rationale for the assessment of the efficacy of VIP as a novel therapeutic approach to the treatment of MS.
Materials and methods
Animals and peptides
Female SJL/J mice 8 weeks old were obtained from Jackson Laboratories (Bar Harbor, ME). PLP139–151 (HCLGKWLGHPDKF) peptide was synthesized using solid phase techniques and HPLC purified by Alpha Diagnostic International (San Antonio, TX). VIP was purchased from Calbiochem (Laufelfingen, Switzerland). The VPAC1-agonist [K15,R16,L27]VIP1-7-GRF8–27 and the VPAC2-agonist Ro 25–1553 were previously described 8.
EAE induction and treatment
Relapsing remitting EAE was induced in SJL/J mice by s.c. immunization with 150 μg of PLP139–151 emulsified in CFA (Difco, Detroit, MI) containing 500 μg of M. tuberculosis H37 RA (Difco). VIP treatment consisted in the administration i.p. of 2 nmol (6.6 μg/mouse/day) on days 5, 7 and 9. For adoptively transferred EAE, DLN cells (5 × 106) were purified 10 days after immunization and restimulated in vitro with 10 μg of PLP139–151 for 72 h, and injected (5 × 106 cells) i.p. into naive SJL/J mice. Animal experimental protocols were reviewed and approved by the Ethical Committee of the Spanish Council of Scientific Research (CSIC).
For CD4+CD25+ T cell depletion, SJL/J mice were treated i.v. with 1 mg anti-CD25 Ab (clone PC61) 24 h before immunization with PLP139–151 as described 28. CD4+CD25+ T cell depletion was >98% in spleen at the time of EAE induction and 72 h later, as determined by flow cytometry.
Mice were scored daily for signs of EAE according to the following clinical scoring system: 0, no clinical signs; 0.5, partial loss of tail tonicity; 1, complete loss of tail tonicity; 2, flaccid tail and abnormal gait; 3, hind leg paralysis; 4, hind leg paralysis with hind body paresis; 5, hind and fore leg paralysis; and 6, death.
Tissue collection and cell isolation
At various time-points after immunization, DLN, brain and spinal cord were removed. Single-cell suspensions were obtained from spleen and DLN. Brain mononuclear cells (BMNC) were isolated by Percoll gradients as described29. For the isolation of different T cell populations (CD4+, CD4+CD25+, CD4+CD25–), DLN cells and BMNC were labeled with PE-anti-CD25 and PerCP-anti-CD4 Ab as described below, and the different populations were gated and sorted using a FACS-Calibur flow cytometer (Becton Dickinson, San Diego, CA). APC were prepared by immunomagnetic T cell depletion of SJL/J spleen cells using microbeads-conjugated anti-CD8 and anti-CD4 mAb (Miltenyi Biotech, Germany), followed by treatment with 50 μg/mL mitomycin C (Sigma). Isolated CD4+CD25– cells (0.5 × 106 cells/ml) were stimulated for different times with anti-CD3 (5 μg/mL) and anti-CD28 (1 μg /mL) Ab in the absence or presence of VIP (10–8 M).
Total RNA was isolated from CD4 T cells or sorted CD4+CD25+ (106 cells) using the Ultraspec RNA reagent (Biotecx, Houston, TX). Two micrograms of total RNA was reverse transcribed with oligo-dT primers and MMLV-RT polymerase (Invitrogen). Quantitative real-time RT-PCR was performed in an ABI PRISM cycler (Applied Biosystems) using a SYBR Green PCR kit from Applied Biosystems. A threshold was set in the linear part of the amplification curve, and the number of cycles needed to reach the threshold was calculated for each gene. Relative mRNA levels were determined by using standard curves for each individual gene and further normalization to HPRT. Melting curves established the purity of the amplified band. Primer sequences used were: Nrp1 (5′-GCCTGCTTTCTTCTCTTGGTTTCA-3′, 5′-GCTCTGGGCACTGGGCTACA-3′); Foxp3 (5′-CTGGCGAAGGGCTCGGTAGTCCT-3′, 5′-CTCCCAGAGCCCATGGCAGAAGT-3′); HPRT (5′-TGGAAAGAATGTCTTGATTGTTGAA-3′, 5′-AGCTTG CAACCTTAACCATTTTG-3′).
Assessment of T cell autoreactive response
DLN cells were recovered from the SJL/J mice at the peak of clinical EAE (day 16 postimmunization). Cells (106 cells/mL) were stimulated in complete medium (RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) with PLP139–151 (10 μM) for 48 h (for cytokine determination) or for 72 h (for proliferative response). Cell proliferation was evaluated by using a cell proliferation assay (BrdU) from Roche Diagnostics GmbH (Mannheim, Germany). Cytokine content in culture supernatants was determined by specific sandwich ELISA5. To determine the suppressive capacity of regulatory CD4 cells, autoreactive CD4 cells (4 × 105 cells/well) isolated from EAE-SJL/J mice were stimulated with spleen APC (105 cells/well) and PLP139–151 (10 μM) in the absence or presence of DLN CD4 cells from untreated or VIP-treated EAE-SJL/J (5 × 104 cells/well). Some cultures were performed in the presence of blocking anti-IL-10 (10 μg/mL), anti-TGF-β1 (40 μg/mL) and/or anti-CTLA4 (10 μg/mL) mAb, or of IL-2 (100 U/mL), all from BD PharMingen. To determine the cell-contact dependence of the regulatory response, we placed EAE rCD4 cells (5 × 104 cells) with spleen APC (105 cells) and PLP139–151 (10 μM) in the bottom well of a Transwell system, and DNL regulatory CD4 cells (2 × 104 cells) isolated from VIP-treated EAE mice with spleen APC (105 cells) and PLP139–151 (10 μM) in the upper Transwell chambers. After 72 h, we measured the proliferative response of the autoreactive CD4 cells from the bottom well.
Flow cytometric analysis
BMNC, DLN cells and spleen cells incubated with various mAb (PE-anti-CD25, FITC-anti-CD62L, FITC-anti-CD69, FITC-anti-GITR, FITC-anti-CD45RB, PerCP-anti-CD4, 2.5 μg/mL final concentration, all from BD PharMingen, FITC-anti-Foxp3 from eBioscience, San Diego, CA) were fixed in 1% paraformaldehyde and analyzed on a FACScalibur flow cytometer. We used isotype-matched Ab as controls, and IgG block (Sigma) to avoid nonspecific binding to Fc-receptors. For analysis of intracellular CTLA-4, BMNC and DLN cells were stained with PerCP-anti-CD4 and FITC-anti-CD25 mAb, fixed with Cytofix/Cytoperm solution (BD PharMingen), incubated with PE-anti-CTLA-4 mAb diluted in 0.5% saponin, and analyzed by flow cytometry.
Immunohistochemistry was performed on adjacent cryosections of spinal cords with biotin-anti-CD4 or biotin-anti-Foxp3 mAb (dilution 1:500) and avidin-peroxidase-DAB staining (Vectastain ABC kit, Vector Laboratories) following manufacturer's recommendations.
Tracing adoptively transferred cells
To trace PLP-specific autoreactive T cells in vivo, spleen and DLN cells from immunized SJL/J mice recovered on day 10 post-immunization were stimulated in vitro with 10 μg PLP139–151 for 72 h, CD4 T cells were isolated as described above and incubated (2 × 107 cells/ml) with 10 mM CFSE (Molecular Probes, Eugene, OR) at 37°C for 20 min. CFSE-labeled cells were injected i.p. (2 × 107 cells/mouse) into naive SJL/J mice. BMNC and DLN cells were isolated 10 days post-transfer and the presence of CFSE-labeled CD4 cells was determined by flow cytometry. Mitotic events were determined as described30.
The Mann-Whitney U-test to compare nonparametric data for statistical significance was applied on all clinical results and cell-culture experiments.
This work was supported by the following grants: NIH (2RO1A047325, DG and MD), Spanish Ministry of Health (PI03/0526, MD), and La Caixa Foundation (NE03–009, MD).
Note added in proof
The manuscript referred to in the text as ‘submitted for publication’, which describes the therapeutic effect of VIP on EAE, has now been accepted for publication. The publication details are