Migration to Local Lymph Nodes Arthritis by Hindering Dendritic Cell ...

Published September 28, 2015, doi:10.4049/jimmunol.1401842 The Journal of Immunology

FTY720 Abrogates Collagen-Induced Arthritis by Hindering Dendritic Cell Migration to Local Lymph Nodes Yanping Han,*,†,‡,1 Xing Li,*,†,1 Qingyou Zhou,*,† Hongyu Jie,*,† Xiaobin Lao,*,† Jiaochan Han,*,† Juan He,*,† Xinxia Liu,‡ Dongsheng Gu,*,†,x Yi He,*,† and Erwei Sun*,† Because dendritic cells (DCs) play critical roles in the pathogenesis of rheumatoid arthritis, modulation of their functions could serve as a novel therapy. In this study, we demonstrated that FTY720 treatment significantly suppressed the incidence and severity of collagen-induced arthritis (CIA) in DBA/1J mice via the modulation of DC functions. In FTY720-treated CIA mice, a decrease in the number of DCs in local draining lymph nodes (LNs) was observed. In vitro, FTY720 inhibited the trafficking of LPS-stimulated bone marrow–derived DCs (BMDCs). Decreased secretion of CCL19 and downregulation of CCR7 on DCs may explain the mechanisms underlying the impairment of DC migration induced by FTY720. In a DC-induced mouse arthritis model, FTY720 treatment also suppressed the incidence and severity of arthritis, which was correlated with a decrease in the migration of injected BMDCs to draining LNs. Although lower levels of costimulatory molecules (CD40, CD80, and CD86) and I-Aq expressed on LN DCs were observed in FTY720-treated mice, in vitro analysis showed no effect of FTY720 on LPS-stimulated BMDC maturation. Furthermore, LN cells from FTY720-treated CIA mice displayed diminished production of proinflammatory cytokines in response to collagen II and Con A stimulation. In addition, the ratio of Th1/Th2 in the draining LNs of mice with DC-induced arthritis was decreased upon FTY720 treatment. This finding was consistent with the fact that FTY720 suppressed IL-12p70 production in cultured BMDCs. Taken together, these results indicate that inhibition of DC migration by FTY720 may provide a novel approach in treating autoimmune diseases such as rheumatoid arthritis. The Journal of Immunology, 2015, 195: 000–000.

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endritic cells (DCs) are powerful APCs and play a central role in the initiation and regulation of immune responses (1–3). After uptake of Ag, DCs are involved in several phases of molecular events to orchestrate an adaptive immune response: Ag processing, antigenic peptide assembly on surface MHC molecules, maturation, and migration to draining lymph nodes (LNs), where they interact with naive T lymphocytes and shape them to be Ag-specific reactive T cells (2). Therefore, DCs are thought to be crucial for triggering many types of diseases such as allergic and autoimmune diseases, including multiple sclerosis and rheumatoid arthritis (RA) (2, 4, 5). Mature DCs were found to be infiltrated in joint fluid and synovial tissue and contributed significantly to the local inflammatory environment of RA (6). DCs in LNs may play more important *Department of Rheumatology and Immunology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China 510630; †Institute of Clinical Immunology, Academy of Orthopedics, Guangdong Province, China 510630; ‡ Hospital of South China Normal University, Guangzhou, Guangdong, China 510631; and xDepartment of Urology, No. 421 Hospital of PLA, Guangzhou, Guangdong, China 510010 1

Y.H. and X.L. contributed equally to this study.

Received for publication July 21, 2014. Accepted for publication August 24, 2015. This work was supported by National Natural Science Foundation of China Grants 81202359, 81273310, and 81471613. Address correspondence and reprint requests to Dr. Erwei Sun, Department of Rheumatology and Immunology, The Third Affiliated Hospital of Southern Medical University, 183 Zhongshan Avenue West, Guangzhou, Guangdong, China, 510630. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BMDC, bone marrow–derived DC; CIA, collageninduced arthritis; CII, type II collagen; DC, dendritic cell; DCIA, DC-induced arthritis; HEV, high endothelial venule; LN, lymph node; pDC, plasmacytoid DC; RA, rheumatoid arthritis; S1P, sphingosine-1-phosphate. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401842

roles in the pathogenesis of RA in that DCs were more mature in draining LNs than were those in synovium (7). Evidence also exists that adoptive transfer of type II collagen (CII)–pulsed mature bone marrow–derived DCs (BMDCs) can induce erosive inflammatory arthritis through migrating to local LNs (8). Therefore, drugs that interfere with DC maturation and migration could constitute a novel therapy for RA. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid metabolite that regulates many cellular processes, including cell survival, cytoskeletal rearrangements, cell migration, and production of cytokines and chemokines (9, 10). S1P is involved in many kinds of diseases, such as atherosclerosis, cancer, asthma, and autoimmune diseases (9, 11, 12). The novel immune-modulator FTY720 is a chemical derivative of myriocin, and its phosphorylated metabolite FTY720-P is a structural homolog of S1P. FTY720 inhibits S1P function through the internalizing of S1P receptors, making cells unresponsive to S1P (13). More recent data indicate that FTY720 can modulate DC trafficking and function (14). FTY720 has been shown to be a useful agent for the prevention of transplant rejection and autoimmune diseases such as multiple sclerosis (15, 16). In mouse model studies, FTY720 is also effective in preventing arthritis development (17, 18). All of the above findings suggest that FTY720 potentially may be a molecule that affects DC function in RA pathogenesis. However, this idea has not been clarified. Therefore, in this study, we investigated the effect of FTY720 on arthritis development and DC functions in mouse arthritis models, to find out whether FTY720 suppressed mouse arthritis through interfering with DC maturation and migration.

Materials and Methods Mice Male DBA/1J mice (6–8 wk old) were purchased from Shanghai Slac Laboratory Animal (Shanghai, China) and maintained under specific

2 pathogen–free conditions in the laboratory animal center of Sun Yat-sen University (Guangzhou, China). The animal experiments were conducted according to the ethical guidelines for animal experiments of Sun Yat-sen University and under experimental license approved by Guangdong, China (No. 00047894 and No. 00053305).

Induction of collagen-induced arthritis Induction of collagen-induced arthritis (CIA) was performed as described elsewhere (19). Briefly, bovine CII solution (2 mg/ml in 0.05 M acetic acid) (Chondrex) was emulsified with an equal volume of CFA containing 1 mg/ml M. tuberculosis (Chondrex). On day 0, 0.1 ml emulsion was injected s.c. at the base of the mouse tail. On day 21, a booster injection (0.1 ml CII emulsified with IFA) was administered near the primary injection site. Mice were subsequently monitored for signs of arthritis every day from the day of the booster injection until the day of sacrifice on day 50.

Clinical and histological assessment of arthritis Arthritis severity was assessed by clinical scores as follows: 0, normal; 1, mild redness and swelling of the ankle or wrist or apparent redness and swelling limited to individual digits; 2, moderate redness and swelling of the ankle or wrist; 3, severe redness and swelling of the entire paw, including digits; 4, maximally inflamed limb with involvement of multiple joints. The clinical scores per mouse were the cumulative value for all paws, with a maximum score of 16 per mouse. For histological assessment, mice hind limbs were removed, fixed in 10% formalin, and decalcified with 5% formic acid. They were embedded in paraffin, and 5-mm slices were prepared. Sections were stained with H&E.

FTY720 ABROGATES CIA BY HINDERING DC TRAFFICKING Cell suspensions were made by filtering the digested fragments through a stainless steel screen. The cell suspension was stained with FITC– anti–mouse CD11c or FITC–anti–mouse CD3/APC–anti–mouse CD19 (BD Pharmingen, CA) and analyzed with a FACScan cytometer equipped with CellQuest software (Becton Dickinson). Absolute cell number was calculated by multiplying the total cell number by the percentage of DCs/T cells/B cells. For sorting of DCs, LN cells or BMDCs were purified using anti–mouse CD11c magnetic microbeads and FcR blocking reagent (Miltenyi Biotec) according to the manufacturer’s instructions. For determination of DC subsets, LN cells were stained with FITC–anti-CD11c (BD Pharmingen) and PE–anti-langerin, or with PE–anti-B220 and APC–anti–PDCA-1 (BioLegend, CA), then analyzed with flow cytometry. To assess the effect of FTY720 on chemokine receptor expression on DCs, nonerythrocyte single-cell suspensions were prepared from mouse blood samples and stained with FITC–anti–mouse CD11c/APC–anti–mouse CCR7 or APC–anti–mouse CCR5/Alexa Fluor 647–anti–mouse CXCR4, then analyzed with FACS. For in vitro study, BMDCs were treated with or without1 mM LPS and 25 nM FTY720 for 24 h. Then cells were harvested for CCR7/CCR5/CXCR4 detection using flow cytometry. In the study of DC maturation in draining LNs, DCs were enriched with anti-CD11c magnetic microbeads and stained with APC–anti–mouse CD11c and PE–anti–mouse CD40/80/86, or with FITC–anti–mouse CD11c and APC–anti–mouse I-Aq (all from BD Pharmingen). For in vitro study of BMDC maturation, the cells were harvested on day 8, sorted, and replated in petri dishes. Thirty minutes before stimulation with 1 mg/ml LPS, the cells were incubated with10 nM or 25 nM FTY720 and continued to be cultured for 24 h. Then the cells were stained with Abs, and flow cytometry was performed as for in vivo studies.

Immunohistochemistry analysis Immunostaining of TNF-a and IL-6 in mouse synovium was performed with peroxidase labeling techniques (20). Briefly, tissue sections were prepared as described in the histological assessment section above. Next, the tissue sections were deparaffinized, preincubated in PBS for 60 min, and then incubated with the primary Ab against mouse TNF-a or IL-6 (Abcam, Hong Kong, China) at 4˚C overnight. After washing in PBS, the sections were incubated for 60 min with HRP-conjugated secondary Ab (DAKO). The sections were stained by immersing in a solution of 0.05% 3,3-diaminobenzidine (Sigma-Aldrich) and 0.01% hydrogen peroxide in 0.05 M Tris (pH 7.4) for 3 min and counterstained with Mayer’s hematoxylin (DAKO). Positive staining was indicated by brownish deposits in a purple background.

Generation of BMDCs BMDCs were generated as described previously (21, 22). Briefly, femurs and tibias of 6- to 8-wk-old male DBA/1 mice were removed and disinfected with 70% ethanol for 2–5 min. Both bone ends were cut, and bone marrow was flushed with PBS. The bone marrow was cultured for 8 d in100-mm-diameter petri dishes (Falcon, Becton Dickinson, Germany). The cells were cultured with RPMI 1640 (Life Technologies, Germany) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 50 mM 2-ME (all from Sigma-Aldrich), and 10% heatinactivated FCS (Hyclone). On day 0, 2 3 106 bone marrow cells per dish were cultured in 10 ml medium containing 400 U/ml GM-CSF (PeproTech). On day 3, another 10 ml fresh medium containing 400 U/ml GM-CSF was added to the plates. On day 6, half of the culture was replaced with fresh 10 ml medium containing 400 U/ml GM-CSF. The cells were harvested on day 8 and purified using anti-CD11c magnetic microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. The cell purity was consistently .95%. In some experiments, cells were pulsed overnight with 1 mg/ml LPS (Escherichia coli 0111:B4; Sigma-Aldrich).

FTY720 administration In the animal study, FTY720 (Selleckchem) was dissolved in sterile saline and filtered through 0.22-mm microfilm. In the FTY720 treatment group, DBA/1 mice were injected i.p. with a dose of 5 mg/kg per day from the day before immunization until the day of sacrifice. In the CIA model group and control group, mice received saline instead.

Flow cytometry To determine DC distribution in blood and LNs and LN cell composition, heparinized blood samples (200 ml) were lysed using 2 ml hypotonic ammonium chloride buffer. Inguinal and popliteal LNs were cut into small fragments and digested with 0.5 mg/ml collagenase A and 0.02 mg/ml DNase I (Roche, Germany) for 30 min at 37˚C with continuous agitation.

In vitro analysis of DC migration Analysis of the effect of FTY720 on DC migration was conducted using a 24-well Transwell (5-mm pore size; Corning, NY) chemotaxis assay. Briefly, on day 8, BMDCs were sorted and divided into four groups, namely, the medium group (400 U/ml GM-CSF), FTY720 group (400 U/ml GM-CSF + 25 nM FTY720), LPS group (400 U/ml GM-CSF + 1 mg/ml LPS), and LPS + FTY720 group (400 U/ml GM-CSF + 1 mg/ml LPS + 25 nM FTY720; FTY720 added 30 min before stimulation with LPS). The cells were cultured for 24 h and then plated in the top chamber (3–4 3 105 BMDC cells per well) in RPMI 1640 medium containing 20 mM HEPES (Sigma-Aldrich). In the bottom chamber, 200 ng/ml CCL19 (PeproTech) in RPMI 1640 medium was added. After incubation for 4 h at 37˚C, the migrated DCs in the bottom chambers were harvested and counted with flow cytometry (23).

Mouse arthritis induced by adoptive transfer of BMDCs Another arthritis model was developed using CII-pulsed BMDCs according to the method described by Leung et al. (8). On day 8 of culture, BMDCs were harvested, purified, and stimulated by overnight incubation with 1 mg/ml LPS and 400 U/ml GM-CSF, and simultaneously pulsed with 50 mg/ml CII. Then 2 3 105 cells in 50 ml PBS were injected s.c. into the hind footpad of male DBA/1 mice in the model group on day 0. Control mice received LPS-stimulated BMDCs that were unpulsed with CII. In the FTY720 group, on day 8 BMDCs were pretreated with 25 nM FTY720 30 min before stimulation with 1 mg/ml LPS and 50 mg/ml CII, and the hind footpads of mice were injected with 2 3 105 FTY720 (25 nM)– treated CII and LPS-pulsed BMDCs. On day 20, mice of the model group and the FTY720 treatment group received a booster injection. Arthritis severity was assessed by clinical scores from the day of cell injection until the day of sacrifice on day 30, as described before (8).

CFSE labeling and in vivo monitoring of BMDCs On day 8 of BMDC culture, three groups of differently treated cells [DC + CII + LPS for the DC-induced arthritis (DCIA) model group, DC + FTY720 + CII + LPS for the FTY720 group, and DC + LPS for the control group] were incubated with prewarmed PBS containing 10 mM CFSE (Invitrogen) for 15 min at 37˚C and washed before use. CFSE-labeled cells (2 3 105) in 50 ml PBS were injected s.c. into each hind footpad of male DBA/1 mice. At 24 h later, mice were sacrificed, and inguinal and popliteal LN cell suspensions were made as described above. CFSE-positive cells were analyzed using flow cytometry (23).

Cytokine and chemokine measurements Both mouse plasma and LN cell supernatants of CIA, control, and FTY720treated groups were assayed for IL-1b, IL-6, TNF-a, IL-17A, IFN-g, and IL-10 levels with the Bio-Plex kit (Bio-Rad Laboratories) and analyzed

The Journal of Immunology with the Bio-Plex manager software (version 4.0). CCL-19 (SigmaAldrich) in mouse plasma, TGF-b (eBioscience) in LN cell culture supernants, and IL-12p70 (R&D Systems, U.S.) in BMDC supernatants were measured with ELISA kits. For detection of cytokine production by LN cells, inguinal and popliteal LN cell suspensions were plated in 24-well plates (1 3 106 cells per milliliter) and restimulated with 50 mg/ml CII for 4 d or with 5 mg/ml Con A (Sigma-Aldrich) for 2 d. The supernatants were collected for cytokine detection. To study the effect of FTY720 on BMDC cytokine production, on day 8 of BMDC culture, the cells were harvested, sorted, and stimulated overnight with 1 mg/ml LPS. FTY720 (1 nM, 5 nM, 10 nM, 25 nM, and 50 nM) was added 30 min before the stimulation of LPS. For the IL-12 signaling mechanism study, 1 mM wortmannin was added to the culture 1 h before the FTY720 treatment. After culture for 24 h, the cell supernatants were collected for the detection of IL-12p70 levels, using ELISA kits (R&D Systems).

Flow cytometric analysis of Th1 and Th2 Mice of the DCIA model group and FTY720 treatment group were sacrificed on day 30. Popliteal and inguinal LN cell suspensions were plated in 24-well plates (1 3 106 cells/ml). The cells were stimulated with 50 ng/ml PMA, 1 mM ionomycin, and 2 mM monensin (all from Sigma-Aldrich) and incubated for 4 h at 37˚C with 5% CO2. For Th1 and Th2 analysis, the cells were stained with FITC–anti–mouse CD4 at 4˚C for 30 min and then stained with PECy5–anti–mouse IFN-g (for Th1 detection) or PE–anti–mouse IL-4 (for Th2 detection) (all the Abs were from BD Pharmingen). The stained cells were analyzed with flow cytometry.

Statistics Statistical analysis was performed using SPSS 16.0 software. All data are expressed as mean 6 SEM. The mean articular index was analyzed using the nonparametric Mann–Whitney U test. The rest of the data were analyzed using one-way ANOVA. The p values , 0.05 were considered significant differences between groups.

Results FTY720 suppressed the development of CIA To know the effect of FTY720 treatment on CIA development, DBA/1 mice were immunized with CII/CFA and treated with FTY720 (5 mg/kg per day). In the model group, clinical signs of arthritis began to develop on day 25 after the first immunization and

FIGURE 1. Effect of FTY720 treatment on CIA. DBA/1 mouse arthritis was induced by an s.c. injection of CII emulsified with CFA at the base of the mouse tail at day 0 and a booster injection at day 21 in the CIA model group (n = 6) and FTY720 treatment group (n = 6). In the FTY720 treatment group, mice were i.p. injected with FTY720 at a dose of 5 mg/kg/d from the day before immunization until the day of sacrifice. Mice of the CIA model group were injected with saline daily. The incidence of arthritis (A) and the mean articular index (B) were monitored every day from the day of booster injection. On day 50, mice were sacrificed to perform histological studies. (C) H&E staining of paw sections of three groups of mice. (Upper lane, original magnification 340; lower lane, magnification 3100.) Results are expressed as mean 6 SEM. All data are representative of three independent experiments. *p , 0.05 for comparisons between the FTY720 treatment group and the CIA model group.

3 progressed to day 38. FTY720 treatment delayed the onset time and reduced the incidence and mean articular scores (Fig. 1A, 1B). Histological examination revealed that FTY720 treatment markedly reduced synovial hyperplasia, inflammatory cell infiltration, and adjacent cartilage and bone erosion, as compared with the nontreated CIA model group (Fig. 1C). We next investigated the levels of some key proinflammatory cytokines, such as TNF-a, IL-1b, IL-6, IL-17, and IFN-g, and an anti-inflammatory cytokine, such as IL-10, in the mouse plasma at different time points (day 20, the day before the booster immunization; day 35, the day on which CIA developed most aggressively; and day 50, the day that CIA reached a plateau) after the first immunization. As shown in Fig. 2A, high concentrations of TNF-a, IL-1b, IL-6, IL-17, and IFN-g were detected in the model group mice, whereas in the FTY720-treated group, all of these proinflammatory cytokines were significantly reduced to the levels found in control mice. In contrast, FTY720 treatment also increased the IL-10 level in mouse plasma as compared with the CIA model group. In RA, the most critical site of cytokine production is the affected joints, particularly within the synovium (24). Hence, we also assessed TNF-a and IL-6 expression in mouse paw sections using immunohistochemistry. The results showed that the expression of TNF-a and IL-6 was also dramatically downregulated by FTY720 treatment (Fig. 2B) FTY720 inhibited DC migration, possibly through downregulation of chemokine and chemokine receptor In previous studies, the effect of FTY720 or other S1P inhibitor that suppresses the development of mouse arthritis was assumed to be the result of the inhibitor’s ability to sequestrate autoimmune T lymphocytes in the thymus or secondary lymph organs, thus blocking the T lymphocytes’ migration to joint tissue and abrogating inflammation (17, 25). Although T cell sequestration may partially contribute to the mechanism by which FTY720 inhibits CIA development, we believe DCs may play an even more fundamental

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FTY720 ABROGATES CIA BY HINDERING DC TRAFFICKING

FIGURE 2. Effect of FTY720 treatment on cytokine production in plasma and synovium. The FTY720 treatment, CIA model, and healthy control (Ctrl) groups of mice were sacrificed on days 20, 35, and 50. (A) Cytokine production in the plasma of the three groups. (B) Immunostaining of TNF-a and IL-6 in synovium (original magnification 3200). Results are expressed as mean 6 SEM. All data are representative of three independent experiments. *p , 0.05 for comparisons between the FTY720 treatment group and the CIA model group.

role in this process, considering that DCs instruct naive T cells to become effector cells. Therefore, in this study we mainly focused on DC functions, especially on DC migration ability and maturation state. To assess the effect of FTY720 on DC migration, we first examined the effect of FTY720 on DC distribution in peripheral blood and draining LNs. Our results showed that FTY720 decreased the absolute number of DCs in inguinal and popliteal LNs at different time points after the first immunization compared with that in the model group, whereas no differences in the proportion and number of blood DCs were observed between those two groups (Fig. 3A). The lower number of DCs in LNs of FTY720-treated mice indicates that FTY720 may inhibit DC migration from peripheral tissues to LNs. To further verify this idea, we cultured BMDCs of DBA/1 mice and conducted an in vitro study in a Transwell system and found that FTY720 blocked migration of mature BMDCs (stimulated by 1 mg/ml LPS) from the top chamber to the bottom chamber in response to CCL19 (Fig. 3C). At the concentration we used in our in vitro studies, FTY720 had no effect on BMDC apoptotic and necrotic death (Supplemental Fig. 1). There are two ways for DCs to migrate to the draining LNs. One is through afferent lymphatic vessels that are used by peripheral tissue–resident DCs such as Langerhans cells in the epidermis. The other is through high endothelial venules (HEV); this is the route for plasmacytoid DCs (pDCs) (26, 27). To elucidate the route by which FTY720 interferes with DC migration to local LNs, we examined DC subsets (langerin+ DCs and pDCs) in the draining LNs. The results showed that in the total DC population

sorted with magnetic microbeads, the proportion of langerin+ DCs was markedly decreased on day 50 in the FTY720-treated group compared with that in the model group (Fig. 3B). Similarly, PDCA-1 and B220 double positive pDCs were also significantly reduced on day 50 after FTY720 treatment. These findings indicate that FTY720 could hinder the traffic of DCs to draining LNs through both afferent lymphatic vessels and blood vascular HEV. The mechanisms underlying the FTY720 inhibitory effect on DC trafficking may be associated with some key chemokines and their receptors that are crucial for DC migration to local LNs. In addition, the results from the above in vitro Transwell study strongly indicate the involvement of chemokine CCL19 and/or its receptor CCR7. We therefore performed both in vivo and in vitro studies to analyze the effect of FTY720 on the level of CCL19 in mouse plasma and the expression of CCR7, CCR5, and CXCR4 on the DC membrane. Results showed that FTY720 treatment decreased the CCL19 level in mouse plasma (Fig. 4C) and downregulated the expression of CCR7 on both mouse blood DCs (Fig. 4A) and in vitro cultured BMDCs (Fig. 4B) but had no effect on CCR5 and CXCR4 expression (data not shown). Thus, modulation of CCR7 expression and CCL19 secretion may contribute to the influence of FTY720 on DC migration. FTY720 suppressed the development of arthritis induced by BMDCs To more directly investigate the role of DC migration in the initiation of arthritis and the effect of FTY720 on this process, we used

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FIGURE 3. Effect of FTY720 treatment on DC trafficking. Single-cell suspensions from blood or inguinal and popliteal LNs of CIA/FTY720 treatment/ healthy control (Ctrl) mice were prepared and stained with FITC- CD11c Ab to assess the proportion and number of DCs. To determine DC subsets in LNs, LN cells were purified using MACS CD11c microbeads and FcR blocking reagent, and stained with FITC–anti-CD11c and PE–anti-langerin, or with PE– anti-B220 and APC–anti–PDCA-1. (A) The percentage and absolute number of DCs in draining LNs and peripheral blood. (B) The percentage of langerin+ DCs and pDCs (PDCA-1 and B220 double positive) in total LN DCs. In vitro study of FTY720 on BMDC migration was conducted using a 24-well Transwell chemotaxis assay. Immature (unstimulated) or mature (stimulated with 1 mg/ml LPS) BMDCs were treated with or without 25 nM FTY720 for 24 h. Then the cells were plated in the top chamber and 200 ng/ml CCL19 in RPMI 1640 was added in the bottom chamber. After 4 h of incubation, migrated DCs in the bottom chamber were harvested and counted by FACS. (C) Migration of FTY720-treated or untreated BMDCs in Transwell assays. Results are expressed as mean 6 SEM. All data are representative of three independent experiments.*p , 0.05, **p , 0.01 for comparisons between the FTY720-treated group and the CIA model group in (A) and (B) or between the LPS + FTY720 group and the LPS group in (C).

a novel mouse arthritis model that is induced by migration of congenic BMDCs into local LNs, as described by Leung et al. (DCIA model) (8). In our experiments, mouse arthritis was induced by the s.c. injection of 2 3 105 CII-pulsed mature BMDCs (stimulated with LPS) into the foot pads of DBA/1 mice. In the model group, arthritis began to develop at day 22 (the third day after a booster injection). In the group injected with FTY720treated BMDCs, however, the incidence and the severity of arthritis were markedly reduced (Fig. 5A, 5B). To monitor DC migration, BMDCs were stained with 10 mM CFSE before injection, and those that migrated to the draining inguinal and popliteal LNs were examined with flow cytometry. The results showed that the number of CFSE-labeled FTY720pretreated BMDCs in the draining LNs were significantly decreased as compared with those cells in the DCIA model group (Fig. 5C, 5D). These findings further support the idea that the inhibitory effect of FTY720 on DCIA may be related to the impairment of BMDC migration from the skin to the draining LNs. FTY720 decreased the mature DCs in LNs by inhibiting migration of mature DCs to local LNs Besides migration, we also evaluated the effect of FTY720 on DC maturation. As shown in Fig. 6A and 6B, DCs from LNs of the FTY720-treated group showed lower expression of costimulatory molecules (CD40, CD80, and CD86) and I-Aq than that in the CIA model group. Although the reduced expression may be caused by a direct inhibition by FTY720 of DC maturation, it

may also result from decreased trafficking of mature DCs from peripheral blood or parenchymal tissues to the draining LNs (Figs. 3A–C, 5C). To find out the reasons behind this phenomenon, we conducted further in vitro studies to evaluate the direct effect of FTY720 on DC maturation. As shown in Fig. 6C, LPS (1 mg/ml) dramatically upregulated the costimulatory molecules and I-Aq; however, FTY720 had no effect on the maturation of BMDCs. Therefore, we believe that the decreased expression of costimulatory molecules and MHC II on DCs in draining LNs in FTY720-treated mice is more likely due to its inhibition of the migration of mature DCs to local LNs. FTY720 changed LN cell composition and cytokine production, as well as attenuated DC function The failure of DCs to migrate to local LNs may result in changed LN cell composition and numbers. Therefore, we analyzed T cells and B cells, the two major types of cells in the draining LNs of mice. Results showed that at different stages of the CIA process (day 20, day 35, and day 50), FTY720 significantly decreased the absolute number of T cells and B cells in the draining LNs (Fig. 7A). Then we assessed immune responses in the LNs of CIA- and FTY720-treated mice. Inguinal and popliteal LN cells were isolated, cultured, and restimulated with 5 mg/ml Con A for 2 d or with CII (50 mg/ml) for 4 d. IL-1b, IL-6, TNF-a, IL-17A, IFN-g, and TGF-b levels in the supernatants were analyzed. As expected, in the CIA model group, those proinflammatory cytokines increased

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FIGURE 4. Effect of FTY720 treatment on chemokine secretion and chemokine receptor expression on DCs. Single-cell suspensions from the blood of CIA/FTY720 treatment/healthy control (Ctrl) mice were prepared and stained with FITC-CD11c and APC-CCR7 Ab to assess the expression of CCR7 on mouse blood DCs. An in vitro study of FTY720 on CCR7 expression was conducted on cultured BMDCs that were treated with or without1 mM LPS and 25 nM FTY720 for 24 h. Then cells were harvested for detection of CCR7 expression using flow cytometry. Mouse plasma was collected on day 20 and day 35 after the first immunization. Plasma CCL19 was analyzed by ELISA. (A) The percentage of CCR7-positive DCs in peripheral blood. (B) Effect of FTY720 on CCR7 expression on cultured BMDCs. (C) Effect of FTY720 on mouse plasma CCL19 production. Results are expressed as mean 6 SEM. All data are representative of three independent experiments. *p , 0.05 for comparisons between the FTY720-treated group and the CIA model group in (A) and (C) or between the LPS + FTY720 group and the LPS group in (B).

dramatically after Con A or CII restimulation, whereas in the FTY720-treated group, they were significantly decreased. In addition, FTY720 treatment could increase TGF-b secretion in the cultured LN cell supernatants, as compared with the CIA model group (Fig. 7B). Because immune potent DCs are very important in immune regulation in local LNs, we next evaluated the functions of DCs affected

by FTY720 in vitro. BMDCs (day 8) were cocultured with 1 nM, 5 nM, 10 nM, 25 nM, and 50 nM FTY720 30 min before LPS stimulation and incubated for 24 h. The supernatants were collected to determine IL-12p70, the driving cytokine that is important in influencing naive T cells toward Th1 cells. Results showed that FTY720 pretreatment (25 nM and 50 nM) attenuated IL-12p70 production by BMDCs, and this attenuation could be reversed by a PI3K inhibitor,

FIGURE 5. Inhibitory effect of FTY720 on DCIA development and on migration of injected BMDCs to draining LNs. DCIA was induced by s.c. injection of 2 3 105 CII and LPS-pulsed BMDCs into the hind footpad of male DBA/1 mice at day 0 and a boost injection at day 20 in the DCIA model group (DC + CII + LPS, n = 10) and the FTY720 treatment group (DC + CII + LPS + FTY720, n = 10). Control mice (Ctrl) (DC + LPS, n = 6) received LPSstimulated BMDCs that were unpulsed with bovine CII. In the FTY720 group, the hind footpad of mice was injected with 2 3 105 FTY720 (25 nM)–treated CII and LPS-pulsed BMDCs. For in vivo monitoring of injected BMDC trafficking, BMDCs were first labeled with 10 mM CFSE for15 min, and then 2 3 105 labeled cells were injected into the hind footpad of male DBA/1 mice. Mice were sacrificed 24 h later, and inguinal and popliteal LN cell suspensions were made. CFSE+ cells were analyzed with flow cytometry. Absolute cell number was calculated by multiplying the total cell number by the percentage of CFSE+ cells. (A and B) Incidence of arthritis and mean articular scores of three groups of mice. (C and D) The proportion (C) and absolute number (D) of migrated CFSE-labeled BMDCs in draining LNs. Results are expressed as mean 6 SEM. All data are representative of three independent experiments.*p , 0.05, **p , 0.01 for comparisons between the FTY720 group and the DCIA group.

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FIGURE 6. Effect of FTY720 treatment on DC maturation. Single-cell suspensions from inguinal and popliteal LNs of CIA/FTY720 treatment/healthy control (Ctrl) mice groups were made and sorted using MACS CD11c microbeads. Suspensions of purified cells were stained with APC–anti-CD11c, PE– anti-CD40/80/86, or with FITC–anti-CD11c and APC–anti–I-Aq, and then assayed with flow cytometry to determine the maturation state of DCs in LNs. For in vitro studies on BMDCs, cells were incubated with10 nM or 25 nM FTY720 30 min before stimulation with 1 mg/ml LPS and then continued to culture for 24 h. Then cells were stained with Abs and assayed with flow cytometry, as for in vivo studies. (A and B) Effect of FTY720 on DC maturation in the LNs in vivo. (C) Effect of FTY720 on BMDC maturation in vitro. Results are expressed as mean 6 SEM. All data are representative of three independent experiments. *p , 0.05 for comparisons between the FTY720 treatment group and the CIA model group in (B).

wortmannin, implying that PI3K is involved in the signaling mechanism by which FTY720 inhibits IL-12 production (Fig. 7C). This reduction of IL-12 production is also consistent with our findings that the ratio of Th1/Th2 in the LNs of FTY720-treated mice was decreased compared with that in nontreated DCIA mice (Fig. 7D).

Discussion RA is an autoimmune disease manifested by the infiltration of synovium with DCs, monocytes, T cells, B cells, neutrophils, and NK cells (28). DCs play a critical role in the initiation and perpetuation of RA (8, 29). Modulation of DC functions by pharmacological agents could be used as a potential therapeutic approach to the prevention and treatment of RA (30). Researchers have tried many ways, such as using immunosuppressants (31), vitamin D receptor agonists (32), or genetic tools (33), to manipulate DCs for immunotherapy and have obtained encouraging results. However, these methods mainly focus on the maturation and immune-stimulating function of DCs; little effort has been made to interfere with DC migration, especially their trafficking into LNs following Ag uptake. In this study, we demonstrated that a novel immunosuppressant, FTY720, could suppress the development of CIA. This therapeutic effect of FTY720 in CIA is probably achieved through modulation of DC migration, as evidenced by several novel findings: 1) FTY720 treatment inhibited the onset and severity of arthritis in the conventional CIA; 2) FTY720 reduced the proportion and total number of DCs in the draining LNs; 3) mice injected with FTY720-treated CII-pulsed BMDCs showed lower incidence and severity of arthritis compared with that in the model DCIA mouse group, and this was associated with the reduction of CFSE-labeled FTY720-treated BMDCs in the draining LNs; 4) both afferent lymphatic vessels and blood HEV pathways of DC migration were abrogated by FTY720, as evidenced by the reduction of langerin+ DCs and pDCs to local LNs; 5) downregulation of mouse plasma CCL19 and expres-

sion of CCR7 on the DC surface may contribute to the inhibitory effect of FTY720 on DC migration; 6) the impaired DC migration to local LNs after FTY720 treatment had a profound influence on LN cell composition and cytokine production; and 7) FTY720 suppressed the immunostimulatory functions of BMDCs. Our results indicate that modulation of DC migration to local LNs may be the novel target for RA treatment. Researchers have demonstrated in several mouse disease models, such as experimental asthma, allergic contact dermatitis, and skin allograft transplantation, that FTY720 impaired DC trafficking to draining LNs and alleviated the clinical signs of the disease or prolonged allograft survival (4, 16, 34). Our data in CIA and DCIA models were consistent with those in previous reports. Some previous studies attributed the suppressing effect of FTY720 on mouse arthritis to its ability to sequestrate autoimmune T lymphocytes in the thymus or secondary lymph organs. To tease out the participation of T cells/B cells, we used a novel DCIA model that was induced by migration of congenic BMDCs into local LNs (8). In vitro FTY720 pretreatment of BMDCs enabled us to abrogate the migration of BMDCs without affecting T cells/B cells or other immune cells, thus eliminating the possibility that FTY720 could influence the distribution of T or B cells in mouse blood and LNs. Therefore, the inhibiting role of FTY720 in DCIA is solely the result of its modulation on DCs. The mechanism underlying the effect of FTY720 on DC trafficking involves chemokines and chemokine receptors that are important for DC migration to draining LNs. We found that FTY720 treatment decreased mouse plasma CCL19 levels and the expression of CCR7 on DCs. In addition, FTY720 inhibited migration of two subsets of DCs, namely, langerin+ DCs and pDCs, which are two representative cells migrating through lymphatic vessels and the blood HEV pathway, respectively, to local LNs. CCR7 is the key receptor that guides peripheral tissue–resident DCs, such as Langerhans cells, to migrate

8


FIGURE 7. Effect of FTY720 on LN cell composition and cytokine production and involvement of attenuated DC function. On days 20, 35, and 50 after the first immunization, single-cell suspensions from inguinal and popliteal LNs of CIA/FTY720 treatment/healthy control (Ctrl) mice were prepared and stained with FITC-CD3/APC-CD19 Abs to assess the proportion and number of T cells/B cells in LNs. On day 50, inguinal and popliteal LN cell suspensions of three groups were restimulated in vitro with 50 mg/ml CII for 4 d or with 5 mg/ml Con A for 2 d. The supernatants were collected for cytokine detection using ELISA. In the study of the effect of FTY720 on BMDC IL-12 production, BMDCs (day 8) were purified and incubated with 1 nM, 5 nM, 10 nM, 25 nM, and 50 nM FTY720 30 min before the stimulation with 1 mg/ml LPS. For the IL-12 signaling mechanism study, 1 mM wortmannin was added to the culture 1 h before the treatment of FTY720. After 24 h of culture, cell supernatants were collected for the detection of IL-12p70 levels, using ELISA kits. (A) The proportion and absolute number of T cells/B cells in LNs at different times after the first immunization. (B) Cytokine concentrations in supernatants of draining LN cells restimulated with Con A and CII. (C) Effect of FTY720 on IL-12 production by BMDCs. (D) Effect of FTY720 on the proportion of Th cell subsets in the LNs of DCIA mice. Inguinal and popliteal LN cells of DCIA and FTY720-treated mice were prepared and assessed for Th1 (CD4 and IFN-g double positive cells) and Th2 (CD4 and IL-4 double positive cells) subsets, using flow cytometry. Results are expressed as mean 6 SEM. All data are representative of three independent experiments. *p , 0.05, **p , 0.01 for comparisons between the FTY720-treated group and the CIA model group in (A) and (B) or between 25 nM or 50 nM FTY720 + LPS–treated BMDCs versus a group of LPS treatment alone in (C) or between the DCIA group and the FTY720 group in (D), #p , 0.05 for comparisons between 25 nM or 50 nM FTY720 1 LPS-treated BMDCs and wortmannin-inhibited group in (C).

through lymphatic vessels into LNs (27). Hence, downregulation of CCR7 may be the reason for FTY720 to block langerin+ DC migration through lymphatic vessels to local LNs. Langerin+ DCs are believed to come from skin, and migration of skin DCs to local draining LNs has been proved to be crucial in cutaneous immunity, such as induction of allergic contact dermatitis (34) and the alloimmune response in skin allograft transplantation (16). Because the CIA model was induced through s.c. injection of emulsified CII in tail skin, skin DCs

may participate in CII uptake in skin and Ag presentation in draining LNs. The pDCs circulate in the blood and migrate to LNs through HEVs to present Ags. They are specialized in modulating the strength and duration of NK, T cell, and B cell responses through releasing cytokines and chemokines. Therefore, pDCs are thought to contribute more to the perpetuation of diseases (35). In RA patients, pDCs observed in LNs showed a relatively more mature state than did those in synovium (7). Therefore, the attenuated

The Journal of Immunology clinical symptom of arthritis in the FTY720-treated group may be, at least in part, owing to the impaired migration of pDCs to local LNs. In the study of DCs in draining LNs, we observed a significant decrease in the proportion of mature DCs in the FTY720-treated group compared with the CIA model group. However, this may not be the result of the direct effect of FTY720 on DC maturation because the in vitro study showed no influence of FTY720 on LPSstimulated BMDC maturation. As our previous results have proved the inhibitory effect of FTY720 on mature DC migration to local LNs, we believe that the reduction in mature DCs in FTY720treated mice may also be the result of the decreased ability of mature DCs to traffic to LNs. Once activated and established in LNs, DCs will upregulate a series of adhesion molecules and the costimulatory molecules to interact with and prime naive T cells (36). The consequence of attenuated DC trafficking to draining LNs by FTY720 treatment may result in decreased levels of T cell/B cell activation, differentiation, and proliferation. Our results proved this idea to be correct in that the number of T cells and B cells in inguinal and popliteal LN cells from the CIA model group increased dramatically and produced more proinflammatory cytokines when restimulated ex vivo with Con A or CII, whereas the number of LN T cells and B cells and the cytokine secretion from FTY720treated mice decreased significantly. These results suggest the possibility that downregulation of DC migration leads to modulation of Ag presentation and T cell/B cell differentiation. These findings are, however, contrary to a previous idea that FTY720 can increase T cell/B cell number in the local LNs as the result of cell sequestration. The different outcome of FTY720 treatment regarding T cell/B cell number in LNs may be caused by different protocols in drug administration. T cell and B cell sequestration and the increase in their number in LNs may be the short-term effect of FTY720, and the long-term treatment will eventually decrease their number, considering the impairment of DC homing to the draining LNs. To further assess whether the contribution of DCs to T cell differentiation was effected by FTY720, BMDCs were analyzed for IL-12, a key cytokine used by the DCs to guide differentiation of naive T cells into Th1 cells (37). Evidence showed that gene silencing of IL-12 in DCs could inhibit autoimmune arthritis (33). Our results demonstrated that FTY720 ($25 nM) markedly suppressed the production of IL-12 in LPS-treated mice. This suppression could be reversed by wortmannin, implicating PI3K involvement in the signaling mechanism. Further experiments demonstrated that the ratio of Th1/Th2 in the LNs of FTY720treated mice was decreased as compared with that in DCIA mice, indicating that FTY720 treatment shifted the Th1/Th2 balance toward a Th2-dominant response. In summary, our studies demonstrated that targeting DC migration by a novel immunosuppressant, FTY720, is highly effective in the treatment of mouse arthritis. It has been known that, in RA patients, DCs are mostly in the mature state in arthritic joints and draining LNs. Our results indicate that using novel small molecules to block the migration of mature DCs may be a promising way to treat RA or other autoimmune diseases. Further investigation into the mechanisms and significance of blocking DC migration might open new doors to future clinical treatment of autoimmune or allergic diseases.

Acknowledgments We thank Yongjian Peng (Animal Laboratory Department of Sun Yat-sen University Cancer Center, Guangzhou, China) for providing technical assistance with animal studies.

9

Disclosures The authors have no financial conflicts of interest.

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