Therapeutic Strategy for Rheumatoid Arthritis by Induction of Myeloid-Derived Suppressor Cells with High Suppressive Potential

Abstract

Combination treatment using fingolimod (FTY720), an immunomodulator, and a pathogenic antigen prevents the progression of glucose-6-phosphate isomerase (GPI)_325-339-induced arthritis. In this study, we focused on myeloid-derived suppressor cells (MDSCs; CD11b⁺Gr-1⁺ cells) and investigated the effects of the combination treatment on these cells. DBA/1J mice with GPI_325-339-induced arthritis were treated using FTY720 and/or GPI_325-339 for five days. The expanded CD11b⁺Gr-1⁺ cell population and its inhibitory potential were examined. The percentage of CD369⁺CD11b⁺Gr-1⁺ cells effectively increased in the combination-treated mice. The inhibitory potential of CD369⁺CD11b⁺Gr-1⁺ cells was higher than that of cells not expressing CD369. Among bone marrow cells, the expression of CD369 in CD11b⁺Gr-1⁺ cells increased following stimulation with granulocyte-macrophage colony-stimulating factor, and the expression of CD11c increased accordingly. The increased CD11c expression indicated a decrease in the potential to suppress T cell proliferation based on the results of the suppression assay. The percentage of CD11c⁻CD369⁺ cells in CD11b⁺Gr-1⁺ cells that were induced by the combination treatment also increased, and these cells tended to have a higher capacity to inhibit T cell proliferation. In conclusion, the combination treatment using FTY720 and the pathogenic antigen effectively induces MDSC, which demonstrates a high potential for suppressing T cell proliferation in the lymph nodes, thereby establishing an immune-tolerant state.

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by multiple types of arthritis and progressive joint destruction. Its symptomatology progresses with repeated relapse and remission, and the resulting severe motor dysfunction reduces the patient’s QOL.^1,2) The treatment basis for RA is an accurate diagnosis at an early stage of onset accompanied by appropriate treatment with methotrexate alone or with other disease-modifying anti-rheumatic drugs (DMARDs) as soon as possible. The therapeutic results of RA treatments have been improved dramatically by the development of biological products such as targeting tumor necrosis factor (TNF)-α, TNF-α receptor, and interleukin (IL)-6 receptor, among others, as well as diagnostic technology. However, biological products have been associated with problems such as 1) high treatment cost, 2) relapse after discontinuation of the drug, and 3) various side effects due to immunosuppressive effects.

We previously reported that combination treatment with fingolimod (FTY720) and a pathogenic antigen shows an excellent therapeutic effect on glucose-6-phosphate isomerase (GPI)_325-339-induced arthritis.³⁾ FTY720 is a synthetic structural analog of myriocin (ISP-I) derived from Isaria sinclairii.^4,5) FTY720 is an immunomodulator that targets the sphingosine 1-phosphate (S1P) receptor. The drug is phosphorylated by sphingosine kinase in vivo, acts as a functional antagonist of the S1P₁ receptor, induces internalization of the receptor, and suppresses lymphocyte egress from the secondary lymphoid tissues.^6–8) T cell apoptosis, anergy, and the expansion of regulatory T cells are induced by the administration of a pathogenic antigen under the isolation of pathogenic lymphocytes to the secondary lymphoid tissue by FTY720.⁹⁾ High IL-10 producing glucocorticoid-induced TNF receptor-family related gene/protein⁺CD25⁻ (or fork-head box protein 3⁻) CD4⁺ T cells increased in the lymph nodes of the combination-treated mice.^10,11) We believe that this combination treatment induces immune tolerance within the secondary lymphoid tissue.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population consisting of myeloid progenitor cells, immature granulocytes, immature macrophages, and immature dendritic cells, characterized by the co-expression of CD11b and Gr-1 in mice, and have immunosuppressive ability.^12,13) MDSCs have two subpopulations: granulocytic MDSCs (CD11b⁺Gr-1^high) and monocytic MDSCs (CD11b⁺Gr-1^int); however, there are still many uncertainties about their inhibitory abilities.¹⁴⁾ MDSCs are known to expand during an imbalanced state of the body, such as cancer, inflammation, infectious diseases, autoimmune diseases, etc.¹⁵⁾ In this study, we investigated the effect of combined treatment with FTY720 and GPI_325-339 on MDSCs for inducing remission in GPI_325-339-induced arthritis.

MATERIALS AND METHODS

Mice and Ethics Statement

DBA/1J mice were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and bred under specific pathogen-free conditions. Mice received γ-ray-irradiated food (CRF-1; Oriental Yeast Co., Ltd., Itabashi-ku, Tokyo, Japan) and distilled water ad libitum. All animal experiments were approved by the Institutional Animal Care Committee of Setsunan University (Approval Nos. K16-16, K17-14, K18-14, K19-18, K20-18, and K21-19) and were performed following the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Throughout the experimental procedures, an effort was made to minimize the number of animals used and their suffering.

Induction of Arthritis

The partial peptide of human GPI_325-339 (IWYINCFGCETHAML) was purchased from Eurofins Genomics Inc. (Ota-ku, Tokyo, Japan). The peptide was dissolved in a small amount of dimethyl sulfoxide and then diluted with sterile phosphate-buffered saline (PBS). The peptide solution and Freund’s complete adjuvant containing Mycobacterium tuberculosis H37Ra (BD Biosciences, Franklin Lakes, NJ, U.S.A.) were mixed in equal volumes and emulsified using a syringe. DBA/1J mice (seven- to nine-week-old males) were immunized intradermally with the emulsion (10 µg/150 µL/mouse) at the base of the tail. Additionally, the mice were intraperitoneally administered with pertussis toxin (EMD Chemicals, Inc., Gibbstown, NJ, U.S.A.) diluted in PBS (200 ng/200 µL/mouse/time) on the day of immunization and two days later.¹⁶⁾

Treatment Regimen and Preparation of Cell Suspension

GPI_325-339-induced arthritis mice were divided into four groups and treated from onset (nine to ten days after immunization) for five days; the FTY720 alone group: mice were orally administered FTY720 dissolved in water (1 mg/kg/approximately 100 µL/mouse, once a day); the GPI_325-339 alone group: mice were intravenously administered GPI_325-339 dissolved in PBS (10 µg/100 µL/mouse, once a day); the FTY720 plus GPI_325-339 combination group: mice were orally administered FTY720 and intravenously administered GPI_325-339 as described above. In the placebo group, mice were administered the vehicle alone.^3,9–11) Inguinal lymph nodes were collected in a petri dish containing 2% fetal bovine serum (FBS)-RPMI 1640, homogenized with glass slides to prepare a cell suspension, and filtered. The cells were hemolyzed as needed with Tris-buffered ammonium chloride (17 mM Tris, 140 mM NH₄Cl, pH 7.2) at 4 °C for 5 min.

Flow Cytometry Analysis and Cell Sorting

The cells were allowed to react with anti CD16/32 monoclonal antibody (mAb) diluted in 2% FBS-PBS to block the non-specific binding of Fc receptors. After incubation at 4 °C for 10 min, the cells were stained with 2% FBS-PBS containing fluorescence-labeled mAbs. After incubation at 4 °C for 30 min, the cells were washed with 2% FBS-PBS and centrifuged (5 min at 400 × g, 4 °C). Next, the cells were resuspended in an appropriate volume of 2% FBS-PBS and filtered. Flow cytometry analysis and cell sorting were performed using BD FACS Aria Fusion (BD Biosciences). When the number of sorted cells was small, samples from several individuals were pooled and analyzed. Antibodies used for cell staining were purified anti-mouse CD16/32 mAb (clone: 93, 1 : 50, BioLegend, Inc., San Diego, CA, U.S.A.), phycoerythrin (PE) or brilliant violet (BV) 421-conjugated anti-mouse CD11b mAb (clone: M1/70, 1 : 200, BioLegend), allophycocyanin (APC) or BV510-conjugated anti-mouse Gr-1 mAb (clone: RB6-8C5, 1 : 200, BioLegend), APC/cyanine 7 (Cy7)-conjugated anti-mouse CD4 mAb (clone: GK1.5, 1 : 300, BioLegend), PE/Cy7-conjugated anti-mouse CD25 mAb (clone: PC61, 1 : 50, BioLegend), PE-conjugated anti-mouse CD369 mAb (clone: RH-1, 1 : 200, BioLegend), and fluorescein isothiocyanate-conjugated anti-mouse CD11c mAb (clone: HL3, 1 : 200, BD Biosciences).

Suppression Assay

CD25⁻CD4⁺ T cells from inguinal lymph nodes, axillary lymph nodes, brachial lymph nodes, and mesenteric lymph nodes of naïve DBA/1J mice (eight- to ten-week-old males) were isolated using fluorescence-activated cell sorting (FACS), and the cells were stained with Carboxyfluorescein succinimidyl ester (CFSE) using CellTrace™ CFSE Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.), according to the manufacturer’s instructions. The sample cells (0.2–0.25, 0.4–0.5, or 0.8–1 × 10⁵ cells) from treated mice and CFSE-labeled CD25⁻CD4⁺ T cells (0.8–1 × 10⁵ cells) suspended in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol, Dynabeads™ Mouse T-Activator CD3/CD28 for T-cell expansion and activation (Thermo Fisher Scientific), and 50 units/mL recombinant mouse IL-2 (rmIL-2, Miltenyi Biotec, Bergisch Gladbach, Germany) were added to 96-well flat-bottom plates (BD Falcon, Franklin Lakes, NJ, U.S.A.) and co-cultured at 37 °C with 5% CO₂ for three days. The cells were then stained with propidium iodide (PI) (1 µM), and the decrease in CFSE fluorescence intensity in CFSE⁺ PI⁻ cells was examined using flow cytometry.

RNA-Sequencing (RNA-Seq)

GPI_325-339-induced arthritis mice were untreated or treated with FTY720 plus GPI_325–339. At the end of the treatment, cells were collected from the inguinal lymph nodes, and CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells were isolated by FACS. RNA-seq was performed as previously described.¹⁷⁾ Cells were lysed in RLT buffer (Qiagen, Hilden, Germany) containing 1% 2-mercaptoethanol. Subsequently, RNA reverse transcription was performed using the SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories, Inc., Moutain View, CA, U.S.A.). cDNA samples were fragmented using the Covaris Focused-ultrasonicator S220, and the cDNA library was created using the Kapa Library preparation kit (KAPA Biosystems, Inc., Wilmington, MA, U.S.A.). cDNA library sequences were analyzed using the IonS5 system (Thermo Scientific). The obtained sequencing results were mapped to the reference genome information (mm9, provided by UCSC) using Tophat2, and unmapped sequences were analyzed again using bowtie2. Normalized FPKM values were provided by Cuffnorm of Cufflinks package (version 2.2.1, Trapnell Lab). The obtained gene expression levels of all genes were depicted on a heat map using the g plots R package (Comprehensive R Archive Network; CRAN, https://cran.ism.ac.jp), amap R package (CRAN), and genefilter R package (Bioconductor, https://www.bioconductor.org). To detect differentially expressed genes (DEGs) in CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells from the combination-treated mice and the untreated mice, we used the exact test of edgeR R package (p < 0.05 was assigned to the DEGs).

Cell Culture of Bone Marrow Cells

Femurs and tibias were collected from naïve DBA/1J mice (six- to nine-week-old males). The muscle and residue tissues around the bone were removed using tweezers. Both ends of the bone were cut using scissors, and the bone marrow fluid was extruded with 2% FBS-RPMI 1640, using a syringe equipped with a needle and it was filtered. The cells were hemolyzed with Tris-buffered ammonium chloride (17 mM Tris, 140 mM NH₄Cl, pH 7.2) at 4 °C for 5 min. The bone marrow cells were suspended in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-mercaptoethanol. Next, 50 mg/mL sodium pyruvate was added to 24-well flat-bottom plates (1 × 10⁶ cells/well), and cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (40 ng/mL, PeproTech, Inc., Rocky Hill, NJ, U.S.A.) at 37 °C with 5% CO₂ for five days.

Statistical Analysis

Statistical analyses were performed using Statcel 3 (OMS Publishing, Saitama, Japan). The significance of the differences was determined using the Tukey–Kramer test for multiple comparisons and the Mann–Whitney U test for two-group comparisons. Statistical significance was set at p < 0.05.

RESULTS

The Percentage of CD11b⁺Gr-1⁺ Cells in the Lymph Nodes Increased with the Combination Treatment

MDSCs play an important role in immune suppression. To investigate whether the combination treatment affected the percentage of CD11b⁺Gr-1⁺ cells in the lymph nodes, GPI_325-339-induced arthritis mice were administered FTY720, GPI_325-339, or both. The percentage of CD11b⁺Gr-1⁺ cells in the inguinal lymph nodes increased significantly in mice treated with the pathogenic antigen, GPI_325-339, and showed a tendency to further increase when the antigen was combined with FTY720 (Figs. 1A, B, p < 0.01). The same result was obtained when the analysis was divided into CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cell populations (Figs. 1A, C, D). Additionally, the percentage of CD11b⁺Gr-1^high cells in FTY720-treated mice was not significant but showed an increasing trend compared to the placebo (Figs. 1A, C).

Fig. 1. Combination Treatment with FTY720 Plus Pathogenic Antigen-Induced CD11b⁺Gr-1⁺ Cells Showing High Inhibitory Potential in Inguinal Lymph Nodes

GPI_325-339-induced arthritis mice were treated with FTY720 and/or GPI_325-339 or left untreated, and cells were collected from the inguinal lymph nodes. (A) Representative flow cytometry dot plots of CD11b and Gr-1 expression in lymph node cells. Bar graph of percentage of (B) CD11b⁺Gr-1^high+int, (C) CD11b⁺Gr-1^high, and (D) CD11b⁺Gr-1^int cells in lymph node cells (n = 4–8). CFSE-labeled CD25⁻CD4⁺ T cells derived from naïve mice and CD11b⁺Gr-1⁺ cells were added to wells and stimulated with Dynabeads Mouse T-activator CD3/CD28 for T cell expansion and activation in the presence of rmIL-2 for three days at 37 °C under 5% CO₂. The decrease in fluorescence intensity of CFSE in CFSE⁺PI⁻ cells was analyzed by flow cytometry. (E) Representative histogram showing CFSE level. (F) Bar graphs represent the percentages of divided T cells (n = 3). The results are shown as mean ± standard deviation (S.D.) and the significance of differences was examined using the Tukey–Kramer test (** p < 0.01, * p < 0.05).

CD11b⁺Gr-1⁺ Cells Induced by the Combination Treatment Demonstrated High Inhibitory Potential

To confirm the inhibitory potential of CD11b⁺Gr-1⁺ cells induced by the combination treatment, a suppression assay was performed. CFSE-labeled CD25⁻CD4⁺ T cells were cultured with CD11b⁺Gr-1⁺ cells derived from combination-treated mice or untreated mice. When the ratio of CD11b⁺Gr-1⁺ cells to CFSE-labeled CD25⁻CD4⁺ T cells was 1 : 2 or 1 : 4, there was no difference in their inhibitory potential, but when the ratio was 1 : 1, CD11b⁺Gr-1⁺ cells derived from the combination-treated mice demonstrated high inhibitory potential towards T cell proliferation (Figs. 1E, F).

Variable Gene Expression in the Combination-Treated Mice- and Untreated Mice-Derived CD11b⁺Gr-1^high or CD11b⁺Gr-1^int Cell Population

RNA-seq was performed to characterize the CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cell populations that were increased by the combination treatment and are shown in the heat map (Fig. 2A). The 1592 and 1081 DEGs in the combination-treated mice and the untreated mice were identified in CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells, respectively. In this study, we first extracted DEGs that coded for the proteins localized on the cell surface and searched for surface markers. Among several DEGs, we focused on CD369, the significance of which has not been previously reported in MDSCs and can be isolated by FACS using available antibodies. Since CD369 was identified as a DEG only in CD11b⁺Gr-1^int cells, a volcano plot showing only this cell population is presented in Fig. 2B. The protein expression levels were examined by flow cytometry analysis. We confirmed that the percentage of CD369 increased in both CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells derived from the combination-treated mice (Figs. 2C, D). Furthermore, the mean fluorescence intensity (MFI) of CD369 increased in CD369-positive CD11b⁺Gr-1^high cells and CD369-positive CD11b⁺Gr-1^int cells, with the differences being significant for the former (Fig. 2E).

Fig. 2. RNA Sequencing

GPI_325-339-induced arthritis mice were treated with FTY720 and GPI_325–339 or left untreated, and CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells were collected from the inguinal lymph nodes. Gene expression levels of CD11b⁺Gr-1^high cells and CD11b⁺Gr-1^int cells were analyzed by RNA-seq. (A) All gene expression profiles were visualized using a heat map (n = 2). (B) Volcano plot of CD11b⁺Gr-1^int cells. (C) Representative flow cytometry dot plots of CD369 expression in CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells, (D) Bar graph of the percentage, and (E) The mean fluorescence intensity (MFI) of CD369 in CD369-positive CD11b⁺Gr-1^high cells and CD369-positive CD11b⁺Gr-1^int cells is shown (n = 8–9). The results are shown as mean ± S.D., and the significance of differences was examined by the Mann–Whitney U test (** p < 0.01, * p < 0.05).

The Cell Population Expressing CD369 in CD11b⁺Gr-1⁺ Cells Induced by the Combination Treatment Demonstrated a High Inhibitory Potential

As shown in Figs. 2C–E, the expression patterns of CD369 in CD11b⁺Gr-1^high and CD11b⁺Gr-1^int cells was similar at the protein level. Total MDSCs were used for subsequent analysis. To confirm the inhibitory potential of CD369⁺CD11b⁺Gr-1⁺ cells induced by the combination treatment, a suppression assay was performed. We compared the inhibitory potential of the CD369⁺CD11b⁺Gr-1⁺ and CD369⁻CD11b⁺Gr-1⁺ cell populations derived from the combination-treated mice toward T cell proliferation (Fig. 3A). When the ratio of CD369-positive or -negative CD11b⁺Gr-1⁺ cells to CFSE-labeled CD25⁻CD4⁺ T cells was 1 : 2, CD369⁺CD11b⁺Gr-1⁺ cells significantly suppressed the proliferation of T cells, more than that by CD369^–CD11b⁺Gr-1⁺ cells (Figs. 3A, B, p < 0.01). This tendency was confirmed even when the ratio was 1 : 4 (Figs. 3A, B). Subsequently, the T cell proliferation inhibitory ability of the CD369⁺CD11b⁺Gr-1⁺ cell population derived from the combination-treated mice and untreated mice was compared, and the inhibitory ability of the combination-treated mice-derived CD369⁺CD11b⁺Gr-1⁺ cells tended to be higher than that of the untreated mice-derived CD369⁺CD11b⁺Gr-1⁺ cells (data not shown).

Fig. 3. CD369⁺CD11b⁺Gr-1⁺ Cells Demonstrate Higher Inhibitory Potential than CD369^–CD11b⁺Gr-1⁺ Cells

GPI_325-339-induced arthritis mice were treated with FTY720 and GPI_325-339, and CD369-positive or -negative CD11b⁺Gr-1⁺ cells were collected from the inguinal lymph nodes. CFSE-labeled CD25⁻CD4⁺ T cells derived from naïve mice and CD369-positive or -negative CD11b⁺Gr-1⁺ cells were added to wells and were stimulated with Dynabeads Mouse T-activator CD3/CD28 for T cell expansion and activation in the presence of rmIL-2 for three days at 37 °C under 5% CO₂. The decrease in fluorescence intensity of CFSE in CFSE⁺PI⁻ cells was analyzed by flow cytometry. (A) Representative histogram showing CFSE level. (B) Bar graphs represent the percentage of divided T cells (n = 3–5). The results are shown as mean ± S.D., and the significance of differences was examined using the Tukey–Kramer test (** p < 0.01).

Expression of CD369 and CD11c Reflected the State of CD11b⁺Gr-1⁺ Cells

To examine changes in the expression of CD369 in CD11b⁺Gr-1⁺ cells associated with activation, bone marrow cells were stimulated with GM-CSF for five days, and flow cytometry analysis was performed. The percentage of CD369⁺ cells in CD11b⁺Gr-1⁺ cells increased particularly on day five of culture (Figs. 4A, B, p < 0.01). Focusing on CD369⁺CD11b⁺Gr-1⁺ cells, the expression of CD11c in the cell population also increased (Figs. 4A, C). The ability of CD11c⁺CD369⁺CD11b⁺Gr-1⁺ cells to suppress T cell proliferation was lower than that of CD11c⁻CD369⁺CD11b⁺Gr-1⁺ cells (Figs. 4D, E). CD369 expression increased with activation, and the inhibitory potential of CD369⁺CD11b⁺Gr-1⁺ cells decreased as CD11c expression increased.

Fig. 4. The Expression of CD369 Increases with Activation, and the Inhibitory Potential Decreases with the Expression of CD11c

Bone marrow cells were stimulated with GM-CSF (40 ng/mL) for three or five days at 37 °C under 5% CO₂, and flow cytometric analysis was performed. (A) Representative flow cytometry dot plots of CD369 and CD11c expression in CD11b⁺Gr-1⁺ cells. (B) Bar graph showing the percentage of CD369⁺ cells in CD11b⁺Gr-1⁺ cells (n = 6–8). (C) Bar graph showing the percentage of CD11c⁺ cells in CD369⁺CD11b⁺Gr-1⁺ cells (n = 6–8). CFSE-labeled CD25⁻CD4⁺ T cells derived from naïve mice and CD11c-positive or -negative CD369⁺CD11b⁺Gr-1⁺ cells were added to wells and were stimulated with Dynabeads Mouse T-activator CD3/CD28 for T cell expansion and activation in the presence of rmIL-2 for three days at 37 °C under 5% CO₂. The decrease in fluorescence intensity of CFSE in CFSE⁺PI⁻ cells was analyzed by flow cytometry. (D) Representative histogram showing CFSE level. (E) Bar graphs represent the percentage of divided T cells (n = 3–5). The results are shown as mean ± S.D., and the significance of differences was examined using the Tukey–Kramer test (** p < 0.01, * p < 0.05).

The Percentage of CD11c⁻CD369⁺ Cells in CD11b⁺Gr-1⁺ Cells Increased in the Inguinal Lymph Nodes of the Combination-Treated Mice

Based on the results of this study, the presence or absence of CD369 and CD11c expression was considered an indicator of the inhibitory ability of CD11b⁺Gr-1⁺ cells. Therefore, to determine if there was a change in the percentage of CD11c⁻CD369⁺ and CD11c⁺CD369⁺ cells in CD11b⁺Gr-1⁺ cells induced by the combination treatment, flow cytometry analysis was performed. There was no difference in the percentage of CD11c⁺CD369⁺ cells in CD11b⁺Gr-1⁺ cells relative to the placebo (Figs. 5A, B) but the percentage of CD11c⁻CD369⁺ cells significantly increased (Figs. 5A, C, p < 0.05). Preliminary studies showed that CD11c⁻CD369⁺CD11b⁺Gr-1⁺ cells, who proportion had increased in vivo, tended to have a higher ability to inhibit T cell proliferation compared to CD11c⁺CD369⁺CD11b⁺Gr-1⁺ cells (Figs. 5D, E). Thus, the results reveal that this combination treatment may create an environment in the inguinal lymph nodes that allows highly suppressive MDSCs to probably increase.

Fig. 5. Combination Treatment with FTY720 Plus Pathogenic Antigen Induced the Expansion of CD11c⁻CD369⁺CD11b⁺Gr-1⁺ Cells in Inguinal Lymph Nodes

GPI_325-339-induced arthritis mice were treated with FTY720 and GPI_325-339 or left untreated, and cells were collected from inguinal lymph nodes at the end of treatment. (A) Representative flow cytometry dot plots of CD11c and CD369 expression in CD11b⁺Gr-1⁺ cells are shown. Bar graph of percentage of (B) CD11c⁺CD369⁺ cells and (C) CD11c⁻CD369⁺ cells in CD11b⁺Gr-1⁺ cells (n = 8). CFSE-labeled CD25⁻CD4⁺ T cells derived from naïve mice and CD11c⁺CD369⁺CD11b⁺Gr-1⁺ cells or CD11c⁻CD369⁺CD11b⁺Gr-1⁺ cells derived from the combination-treated mice were added to wells and were stimulated with Dynabeads Mouse T-activator CD3/CD28 for T cell expansion and activation in the presence of rmIL-2 for three days at 37 °C under 5% CO₂. The decrease in fluorescence intensity of CFSE in CFSE⁺PI⁻ cells was analyzed by flow cytometry. (D) Representative histogram showing CFSE level. (E) Bar graphs represent the percentage of divided T cells (n = 3–5). The results are shown as mean ± S.D., and the significance of differences was examined using the Mann–Whitney U test ((B), (C): * p < 0.05) and Tukey–Kramer test ((E): * p < 0.05).

DISCUSSION

Induction of immune tolerance is very important for long-term remission of autoimmune diseases, such as rheumatoid arthritis. The combination of FTY720 and pathogenic antigens may be a successful therapeutic approach that can effectively induce immune tolerance. In the combination treatment, the mobilization of lymphocytes to the local site of inflammation was suppressed mainly by the action of FTY720. Thus, a state in which the pathogenic lymphocytes are isolated from the lymph nodes is created. We believe that exposure to pathogenic antigens under these conditions provokes various immune responses within the lymph nodes and induces immune tolerance.

MDSCs with immunosuppressive activity are characterized by the co-expression of CD11b and Gr-1 in mice.^12,13) Liu et al.¹⁸⁾ reported that the S1P₁-mammalian target of rapamycin (mTOR) signaling pathway negatively regulates MDSC recruitment and function in a mouse model of hepatic injury. As shown in our current study, CD11b⁺Gr-1^high cells showed an increasing tendency in FTY720-treated mice. Thus, we assumed that FTY720 affects the S1P₁ receptor-mTOR signaling pathway by inducing the internalization of the S1P₁ receptor on CD11b⁺Gr-1⁺ cells, contributing to the expansion of MDSCs. A significant increase in CD11b⁺Gr-1⁺ cells was observed in antigen-treated mice, especially in mice co-administered with FTY720. Therefore, it was assumed that MDSCs increased due to the exposure to pathogenic antigens and that its action was enhanced by the synergistic effect of FTY720.

In this study, we found that our combination treatment not only increased CD11b⁺Gr-1⁺ cells in the inguinal lymph nodes but also enhanced their inhibitory potential. RNA-seq analysis revealed that MDSCs induced by the combination treatment highly expressed CD369. CD369, also known as dendritic cell-associated C-type lectin 1 (dectin-1), C-type lectin domain family 7 member A, or β-glucan receptor, is highly expressed in populations of myeloid cells (including monocytes, neutrophils, and macrophages).^19–21) CD369 is also known to be expressed in MDSCs,²²⁾ but its significance has not yet been clarified. Taylor et al.²¹⁾ reported that inflammatory macrophages highly express β-glucan receptors on the cell surface, indicating the role of this receptor in immune surveillance. Since it has been reported that tumor progression is delayed in dectin-1-deficient mice,²³⁾ we considered that MDSCs are involved in the immunosuppressive effect and decided to focus on CD369 in this study. Therefore, when bone marrow cells derived from femurs and tibias were cultured under GM-CSF stimulation and the expression of CD369 was confirmed in the process of inducing differentiation of in vitro MDSCs, it was confirmed that the expression of CD369 increased daily. Thus, we hypothesized that the expression of CD369 increases under the activation by GM-CSF. Similarly, in this study, the expression of CD11c was also increased under the activation by GM-CSF. Most of the CD11c⁺ cells expressed CD369, which suggests that the expression of CD369 is elevated in cell populations containing cells capable of differentiating into CD11c⁺ cells (dendritic cells) rather than as an indicator of MDSCs with high suppressive capacity. Tian et al.²²⁾ reported that treatment with β-glucan, a ligand for dectin-1, promoted the differentiation of monocytic (M)-MDSCs into CD11c⁺F4/80⁺Ly6C^low cells and reduced inhibitory potential towards T cell proliferation. In this study, we confirmed in in vitro CD11b⁺Gr-1⁺ cells that the cells expressing CD11c had lower inhibitory potential towards T cell proliferation than that of cells not expressing CD11c. Although the expression of CD369 in CD11b⁺Gr-1⁺ cells increased due to the combination treatment, the expression of CD11c possibly increased via CD369 signaling and differentiation was promoted in a cell population that had a low potential to suppress T cell proliferation. However, in the inguinal lymph nodes of combination-treated mice, CD369⁺CD11b⁺Gr-1⁺ cells showing low expression of CD11c increased. Vimentin, which is secreted by activated macrophages, is known to be an endogenous ligand for dectin-1.^24,25) However, Lu et al.²⁶⁾ reported that FTY720 reduces the expression of vimentin in cholangiocarcinoma cells owing to its inhibitory effect on the signal transducer and activator of transcription 3 (STAT3) pathway. Therefore, FTY720 may affect the secretion of vimentin, and as a result, the increase in the expression of CD11c mediated by the CD369 signal is regulated. Additionally, Mor-Vaknin et al.²⁴⁾ showed that IL-10, which inhibits protein kinase C activity, blocks the secretion of vimentin. As stated earlier, we have previously shown that this combination treatment increases the IL-10-producing T cell population in the inguinal lymph nodes.¹¹⁾ Therefore, this was also considered one of the CD369 signal-regulated factors.

In conclusion, combination treatment with FTY720 and a pathogenic antigen increased the proportion of MDSCs with high inhibitory capacity towards T cell proliferation in secondary lymphoid tissues. We believe that this MDSC contributes to immune tolerance that can be induced by this combination treatment. In the future, the reason and mechanism underlying the increase of MDSCs and up-regulation of the inhibitory ability of MDSCs must be considered.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (C) (19K08897) from the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

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