Tolerogenic Dendritic Cells Induced by BD750 Ameliorate Proinflammatory T cell Responses and Experimental Autoimmune Encephalitis in Mice
BD750, a novel JAK3/STAT5 inhibitor, can inhibit T cell proliferation. This study aims to evaluate whether BD750 can induce tolerogenic dendritic cells (tolDC) and their function in experimental autoimmune encephalitis (EAE) in mice. Following BD750 treatment, lipopolysaccharide (LPS)-induced maturation of DCs, allogeneic T cell proliferation, Th1 and Th17 cell functional differentiation, and STAT5 and AKT activation were determined. The effect of tolDC loaded with antigen peptide on the development and severity of EAE and splenic Th1 and Th17 cell responses was determined. In comparison with LPS-induced mature DCs (mDCs), BD750 treatment induced tolDC with lower expression levels of costimulatory molecules and proinflammatory cytokines and lower levels of STAT5 phosphorylation. TolDC inhibited allogeneic T cell proliferation and reduced Th1 and Th17 responses. Adoptive transfer of tolDC loaded with myelin oligodendrocyte glycoprotein35-55 inhibited the development and severity of EAE in mice, accompanied by reduced numbers of inflammatory infiltrates and decreased levels of demyelination in the spinal cord tissues. In addition, treatment with tolDC loaded with antigen peptide also significantly reduced the frequency of splenic Th1 and Th17 cells in EAE mice. The effects of tolDC were similar to that of JAK/STAT inhibitor CP690550-treated DCs. In conclusion, treatment with BD750 induced tolDC that inhibited proinflammatory T cell immunity in vitro and in vivo. BD750 and tolDC may be valuable for development of new therapies for EAE and other autoimmune diseases.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease in the central nervous system (CNS), predominantly mediated by autoreactive CD4+ Th1 and Th17 cells. During the pathogenic process of MS, autoreactive CD4+ T cells pass through the blood-brain barrier and migrate into the brain and spinal cord to cause demyelination in the CNS (1,2). Together with defects in the function of regulatory T cells (Tregs) in patients with MS, this enhances pathogenic Th1 and Th17 responses within the brain (3). Therefore, reestablishing antigen-specific tolerance will be of great significance in managing patients with MS (4).
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) and can induce naïve T cell activation. Mature DCs present peptide of antigens by major histocompatibility complex (MHC) molecules and express high levels of costimulatory molecules, such as CD80, CD83, CD86 and CD40, on their surface to provide two necessary signals for full T cell activation. Inflammatory DCs can secrete IL-12, IL-6, TGF-β and IL-23, which promote Th1 or Th17 polarization (5). The levels of proinflammatory cytokines produced by DCs largely depend on the maturation status, ranging from fully mature immunogenic DCs to tolerogenic immature DCs, spanning a wide variety of intermediate semi-mature states. Tolerogenic DCs (tolDC) are usually resistant to maturation, with lower levels of costimulatory molecule expression and inflammatory cytokine production, but higher levels of inhibitory molecule expression. tolDC can induce anergy and apoptosis of effector T cells, but promote Treg responses (6,7). Adoptive transfer of tolDC is a new strategy to restore immune tolerance and may be a therapeutic strategy for autoimmune diseases (8,9). Treatment with type II collagen-pulsed tolDC (dexamethasone/vitamin D3-modified) decreases the percentages of pathogenic Th17 cells in arthritic mice and reduces the severity and progression of arthritis (10). Furthermore, H471-94 peptide-loaded tolDC augments suppressive function of Tregs, suppresses Th17 cells and benefits patients with lupus (11). In addition, tolDC generated in vitro inhibit the process of experimental autoimmune encephalitis (EAE) in mice (12). More importantly, tolDC from relapsing-remitting MS patients can induce stable antigen-specific hyporesponsiveness in myelin-reactive T cells (13).
Induction of tolDC has been experimentally achieved by suppressive agents, including IL-10 and TGF-β; immunomodulatory drugs, such as dexamethasone and vitamin D; and genetic modification (14). Our previous study identified that a benzothiazole derivative, BD750 [2-(2-benzothiazoleyl)-4,5,6,7-tetrahydro-2H-indazol-3-ol, C14H13N3OS, MW: 271.3], is an inhibitor of JAK3/STAT5 signaling and can inhibit T cell proliferation (15). JAK3 is crucial for the maturation of DCs, and JAK3−/− DCs fail to induce T cell proliferation (16,17). STAT5 is crucial for thymic stromal lymphopoietin-dependent DC activation and can upregulate the expression of costimulatory molecules and chemokines (18). Accordingly, we hypothesize that BD750 may induce tolDC, which may inhibit antigen-specific Th1 and Th17 responses and the pathogenic process of EAE in mice. In the current study, we tested the effect of BD750 on the maturation and function of DCs in vitro and the impact of adoptive transfer of BD750-treated DCs on Th1 and Th17 responses in EAE mice. We found that BD750 induced tolDC that impaired allogenic antigen-stimulated T cell responses in vitro. Furthermore, adoptive transfer of tolDC that had been loaded with antigen peptide inhibited Th1 and Th17 responses and the development of EAE in mice. Our findings may aid in the design of new therapies for MS.
Materials and Methods
Animals and Animal Models
C57BL/6 and BALB/c mice were purchased from Dashuo Biotechnology (Chengdu, China). The experimental protocols were approved by the Animal Care and Use Committee at Chengdu Medical College. All mice were bred and maintained in a specific pathogen-free facility in the Experimental Animal Center of Chengdu Medical College at consistent room temperatures (20–26°C) and humidity (approximately 40–70%), with a 12 h/12 h light/dark cycle.
Female C57BL/6 mice (10 wks old, 19–21 g) were used for the experiments aimed at investigating the therapeutic effect of BD750-treated DCs on the development and progression of EAE. Animals were randomized by simple randomization into seven groups (n = 8 mice per group). The mice were immunized subcutaneously at the dorsal flanks with 200 µg myelin oligodendrocyte glycoprotein (MOG)35-55 peptide (China-Peptides, Shanghai, China) emulsified 1:1 with complete Freund’s adjuvant (CFA) supplemented with 4 mg/mL heat-killed Mycobacterium tuberculosis (Chondrex, Redmond, WA, USA). Individual mice were injected intraperitoneally with 500 ng pertussis toxin (PTX) on d 0 and 2. One group of mice received adjuvant and PTX only and served as the control. The mice were monitored daily for clinical symptoms, which were scored as: 0, no clinical signs; 1, paralyzed tail; 2, loss of coordinated movement, hind limb paresis; 3, both hind limbs paralyzed; 4, forelimbs paralyzed; and 5, moribund (19).
Generation of Murine Bone Marrow-Derived Dendritic Cells
Murine bone marrow-derived dendritic cells (BMDCs) were isolated from female C57BL/6 mice (6 wks old, 16–18 g), as described previously (20). Briefly, the animals were anesthetized with intraperitoneal injection of sodium pentobarbital and euthanized by cervical disconnection. Subsequently, their femur and tibia bones were cut with scissors and the marrow was flushed out, followed by passing through a nylon mesh to remove small pieces of bone and debris. The resulting single cells (1 × 106 cells/dish) were cultured in 10% fetal calf serum RPMI 1640 (complete medium) for 4 h. The suspended cells were removed and the adherent cells were cultured in complete medium containing 20 ng/mL of recombinant granulocyte-macrophage colony-stimulating factor and 10 ng/mL of IL-4 (PeproTech) for 6 d. The cells were exposed to fresh medium every 3 d. On d 6 post-incubation, the cells were harvested and some cells were stained with fluorescent-labeled antibodies, followed by flow cytometry analysis. The CD11c+ immature DCs in the remaining cells were purified by magnetic anti-CD11c beads (Miltenyi). The purified immature DCs were pretreated with BD750 or vehicle dimethyl sulfoxide for 12 h, ensuring that the dimethyl sulfoxide was <0.025%. The cells were then stimulated with 100 ng/mL lipopolysaccharide (LPS) to induce DC maturation for 24 h.
Allogeneic Mixed Leukocyte Reaction
To test the effect of BD750-treated DCs on stimulating T cell proliferation, naïve splenic CD4+CD69− T cells were isolated from female BALB/c mice (6 wks old, 17–19 g) using a T cell isolation kit (Miltenyi) and labeled with carboxyfluorescein diacetate succinimidyl ester (1.2 µM; Invitrogen). The naïve T cells were cocultured in triplicate with allogeneic BD750 pretreated DCs (BD750-mDC) at three DC:T cell ratios (1:10, 1:30, 1:100) for 72 h. The proliferation of activated T cells was analyzed by flow cytometry.
The mature DCs were stained in duplicate with fluorescent-labeled antibodies against CD11c, CD69, CD83, CD40, CD80, CD86, MHC II or isotype-matched controls (BioLegend, San Diego, CA, USA). The maturation status of DCs was analyzed by flow cytometry. Some mature DCs were analyzed for their apoptosis by flow cytometry using the PI Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturers’ instruction.
In addition, the functional phenotypes of CD4+ T cells were analyzed by flow cytometry. Briefly, after mixed leukocyte reaction (MLR) coculture, CD4+ T cells were separated from the cell culture, then treated with anti-CD16/32 (Mouse BD Fc Block™), stained with fluorescein isothiocyanate-anti-CD4, fixed, permeabilized and intracellularly stained with PE-conjugated anti-IFN-γ and Alexa Fluor® 647-conjugated anti-IL-17, followed by flow cytometry. Moreover, Tregs were analyzed by flow cytometry using the Transcription Factor Buffer Set (BD Pharmingen), after being stained with APC-anti-CD25 and intracellularly with PE-anti-Foxp3, according to the manufacturer’s instructions. All flow cytometry was performed in the Accuri™ C6 (BD Biosciences) or Cytomics FC 500 (Beckman Coulter).
Enzyme-Linked Immunosorbent Assay
The supernatants of cultured DCs or DCs cocultured with T cells were harvested and the levels of IL-6, IL-10, IL-12, TNF-α, IL-1β, IL-23, IFN-γ and IL-17 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using specific kits (eBioscience), according to the manufacturer’s instructions. Briefly, the control and experimental samples were simultaneously tested in triplicate, and the plates were read at 450 nm (Powerwave XS, BioTek, Winooski, VT, USA).
The purified immature DCs were pretreated with BD750 (1, 5 or 20 µM), CP690550 (1 µM), LY294002 (1 µM) or vehicle for 12 h. The cells were stimulated with 100 ng/mL LPS to induce DC maturation for 24 h. The harvested mature DCs were lysed in radioimmunoprecipitation assay buffer (Millipore), and the protein concentrations in the lysates were determined using a BCA Protein Assay Kit (BioTek). Individual cell lysate samples (20 µg/lane) were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After being blocked with 5% bovine serum albumin, the membranes were incubated with specific antibodies against STAT5, phospho-STAT5, Akt, phospho-Akt or GAPDH (Cell Signaling Technology). The bound antibodies were detected with horseradish peroxidase-conjugated second antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA) and visualized by enhanced chemiluminescence (Millipore). The relative levels of target proteins were normalized to the internal GAPDH and analyzed using ImageJ software.
Adoptive Transfer Experiments
Immature DCs at 6 d post-culture were pretreated with BD750 (20 µM) for 12 h. The cells were pulsed with MOG35-55 (20 µg/mL) for 24 h and subsequently treated with LPS (100 ng/mL) to induce DC maturation for 24 h. The cells were harvested and injected intravenously into mice at −2, 2 and 6 or 7, 11 and 15 d post first PTX injection (1 × 106 cells/mouse). The control mice were injected with PBS.
At 22 d post-induction, the mice were anesthetized with intraperitoneal injection of sodium pentobarbital and perfused with PBS (pH 7.4), followed by 4% (w/v) paraformaldehyde. Spinal cord samples were then fixed in 4% (w/v) paraformaldehyde overnight and paraffin-embedded. The coronal sections (5 µm) of cervicothoracic spinal cord tissues were stained with hematoxylin and eosin and examined for cellular infiltration or with Luxol fast blue/periodic acid-Schiff to determine demyelination.
The data are expressed as mean ± standard error of the mean. The differences among the groups were evaluated using analysis of variance and post hoc least significant different t test or Kruskal-Wallis test using PRISM 6.0 software (GraphPad Software, San Diego, CA, USA). A P value < 0.05 was considered statistically significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
BD750 Treatment Induces tolDC
BD750-Treated DC Functionally Impairs Allogenic T Cell Responses In Vitro
Adoptive Transfer of BD750-Treated DC Inhibits the Development and Reduces the Severity of EAE in Mice
DCs are the most powerful APCs and induce antigen-specific T cell responses (22,23). Mature DCs are powerful for inducing T cell activation, and immature DCs usually promote T cell tolerance. Previous studies have shown that tolDC can inhibit autoimmune diseases, such as rheumatoid arthritis and EAE, in rodents (24,25). Indeed, pharmacological modification is a new strategy to induce tolDC (10,26). It is well known that the JAK/STAT pathway is crucial for the maturation and tolerance of DCs (16). Inhibition of STAT5 can impair the maturation of monocyte-derived DCs with downregulated expression of costimulatory molecules and reduced secretion of IL-12 (27,28). Our previous study identified that BD750 is an inhibitor of JAK3/STAT5 and can inhibit T cell proliferation (15). In this study, we found that BD750 induced tolDC. Evidentially, BD750-treated DCs displayed typically immature morphology. Second, BD750-treated DCs had lower levels of costimulatory molecule and MHC II expression and secreted lower levels of proinflammatory cytokines following LPS activation in vitro. Furthermore, BD750-treaed DCs impaired allogeneic antigen-stimulated T cell proliferation, accompanied by lower frequency of proinflammatory Th1 and Th17 cells and STAT5 phosphorylation. Its inhibitory effect was similar to that of CP690550. Given that tolDC are important to induce T cell tolerance, BD750 may be a valuable agent for inducing T cell tolerance. Notably, BD750 inhibited STAT5 activation but not AKT phosphorylation, consistent with our previous findings (15). This observation suggests that BD750 may be a specific inhibitor of STAT5. We are interested in further examining the molecular mechanisms underlying the action of BD750 in regulating T cell immunity.
Myelin-specific Th1 and Th17 cells are responsible for the pathogenesis of EAE and MS. To understand the potential regulatory role of BD750-induced tolDC, we adoptively transferred antigen peptide-loaded tolDC during the development of EAE in mice. We found that adoptive transfer with antigen peptide-loaded tolDC, particularly at the early stage of inducing EAE, significantly delayed the development of EAE and reduced its severity in mice. Furthermore, treatment with tolDC also reduced inflammatory infiltrates and the degree of demyelination in the spinal cords of mice. Given that tolDC are relatively stable in phenotype and function (13), autologous tolDC may be valuable for inducing T cell tolerance (29). Although it might be difficult for tolDC to pass the CNS (26), they did inhibit splenic Th1 and Th17 responses in EAE mice, similar to a previous report (26). In addition, although BD750 did not affect IL-10 production by LPS-stimulated DCs, BD750-DC secreted a low level of IL-12. A previous study reported that decreased IL-12 production of tolDC plays an important role in inducing Treg differentiation (25), so BD750-DC secreted a low level of IL-12, which could enhance tolDC-mediated Tregs differentiation in MLR and subsequently expand the Treg population.
tolDC induced T cell tolerance, dependent on antigen peptide loading. Therefore, tolDC loaded with antigen peptide may be promising for inhibiting T cell autoimmunity and EAE progression. Our data indicate that adoptive transfer with antigen peptide-loaded tolDC should start at the early stage of the disease process. We speculate that this was because antigen-specific T cell response in the CNS was possibly the first demyelinating event. Similarly, we found that continual transfer of tolDC loaded with antigen peptide was necessary for the control of disease progression. Because MS is a chronic disease, it is also very difficult for us to speculate the long-term efficacy of passive transfer therapy and how frequently therapy is necessary to control disease progression. Therefore, further investigations are warranted to determine the safety and effectiveness of tolDC-based immunotherapies for MS.
Our data indicate that BD750 induced tolDC by downregulating their costimulatory molecule expression and cytokine production. BD750-treated DCs impaired allogeneic antigen-stimulated T cell proliferation. BD750-modified DCs induced hyporesponsiveness and impaired proinflammatory cytokine secretion by allogeneic T cells in MLR. In addition, the tolDC pulsed with MOG35-55 effectively attenuated autoantigen-specific Th1/Th17 differentiation, and adoptive transfer of tolDC loaded with antigen peptide inhibited autoimmune responses in a mouse model of MS. Therefore, our findings may provide new insights into inducible immune tolerance and aid in the design of new therapies for MS.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript. This work was supported by grants from the National Natural Science Foundation of China (nos. 81202363, 81302786, 81273530), the Research Fund of Development and Regeneration Key Laboratory of Sichuan Province (no. SYS16-002), the Program for Provincial Universities Innovative Research Team in Sichuan Province (no. 13TD0028), the Innovative Research Team Fund of Chengdu Medical College (no. CYTD15-01), the Scientific Research Fund of Sichuan Provincial Education Department (no. 16ZB0271) and the National Undergraduates Innovating Experimentation Project (no. 201513705005).
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