THU0085 RESOLVIN D5 MODULATES TH17/TREG CELL DIFFERENTIATION AND SUPPRESSES OSTEOCLASTOGENESIS

Resolution phase of acute inflammation has been recognized not passive but active process during the last 2 decades. This active process is highly regulated by novel families of potent bioactive lipid mediators, which are coined as specialized proresolving mediators (SPMs) including resolvins. Little has been known, however, about how resolvins are involved in chronic inflammation, such as rheumatoid arthritis (RA).To investigate whether lipid mediators (LM) are involved in the pathogenesis of RA.We investigated lipid mediator profiling in the paws of SKG arthritis mice by using lipid chromatography (LC) /mass spectrometry (MS) /MS-based LM metabololipidomics. CD4+T cells from spleens of SKG mice were cultured on anti-CD3/ CD28Abs precoated plate with IL-6/TGF-β, anti-IFNγ/IL-4 and analyzed by flow cytometry. CD4+T cells were labeled with CFSE, and cell proliferation was analyzed by flow cytometry. Mouse bone marrow cells were cultured with M-CSF and RANKL, and TRAP-positive multinucleated cells were defined as osteoclasts. Osteoclast differentiation markers were analyzed by qRT-PCR. RvD5 or normal saline was administered daily into the peritoneal cavity of arthritic SKG mice.RvE3, RvD1, RvD3, RvD5 and Maresin2 were significantly elevated on the paws of arthritic SKG mice. Among the elevated SPMs, only RvD5 levels on arthritic paws were significantly correlated with arthritis disease activity (Figure 1). We demonstrated that RvD5 suppressed Th17 cell differentiation, and facilitated Treg cell differentiationin vitro. In addition, RvD5 inhibited CD4+T cell proliferation. Furthermore, RvD5 attenuated osteoclast differentiation (Figure 2) and interfered osteoclastogenesis at the molecular level. In thein vivoexperiment, incidence of arthritis tended to be lower in RvD5-treated mice than that in control group, although there was no significant difference.RvD5 is increased in the paws of arthritic mice, and that RvD5 suppresses Th17 cell differentiation and CD4+T cell proliferation, facilitates Treg cell differentiation, and suppresses osteoclastogenesis.[1]Serhan, C. N. 2014. Pro-resolving lipid mediators are leads for resolution physiology. Nature 510: 92-101.[2]Buckley, C. D., Gilroy, D. W., Serhan, C. N. 2014. Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity 40: 315-27.[3]Colas, R. A., Shinohara, M., Dalli, J., Chiang, N., Serhan, C. N. 2014. Identification and signature profiles for pro-resolving and inflammatory lipid mediators in human tissue. Am J Physiol Cell Physiol 307: C39-54.The authors thank Shino Natsui (Department Rheumatology and Clinical Immunology, Kobe University Graduate School of Medicine) for providing technical assistance.None declared


Introduction
Over the last two decades, it has become increasingly clear that resolution of acute in ammation is not a passive process, but requires active modulation. This is tightly regulated by families of novel potent bioactive LMs, which have been termed "specialized pro-resolving mediators" (SPMs) 1,2 . These are synthesized from in ammatory exudate and stimulate the resolution phase via enhancement of neutrophil apoptosis, macrophage in ltration, M2-type polarization and efferocytosis to return to the baseline homeostatic state 3 . When acute in ammation fails to resolve appropriately and leads to chronic in ammation, it is thought that dysregulation of these SPMs may be involved in chronic disorders such as rheumatoid arthritis (RA). RA is a chronic in ammatory disease characterized by synovial in ammation and hyperplasia, autoantibody production, and bone destruction. These features are caused by abnormalities in adaptive and innate immune processes. Interactions between leukocytes, synovial broblasts and OCs drive the chronic phase in the pathogenesis of RA 4 .
The effectiveness of n-3 polyunsaturated fatty acid supplementation for treating RA has been reported 5 .
More recent randomized clinical trials indicated that docosahexaenoic acid (DHA) supplementation tended to ameliorate RA disease activity 6 . Of the SPMs, RvD1 and RvD3, both of which are derived from DHA, have been reported to reduce arthritis severity in a K/BxN serum transfer arthritis model, and to be detectable in the synovial uid of RA patients 7,8 . Other SPMs (RvD5, Maresin 1 (MaR1) and LipoxinA 4 ) have also been identi ed in synovial uid in RA patients 9 . According to one report, MaR1 decreased arthritis and joint injury scores in a collagen-induced arthritis model 10 .
Several SPMs have been reported to have regulatory effects on the cellular targets that establish the pathology of RA. RvD1, RvD2 and MaR1 suppress Th1 and Th17 polarization and facilitate regulatory T cells (Treg) differentiation of naïve CD4 + T cells isolated from human peripheral blood mononuclear cells 11 . The ratio of Treg/Th17 cells is elevated in lymph nodes from MaR1-treated collagen-induced arthritis mice 10 . In addition, RvD1 and RvE1 have been shown to inhibit osteoclastogenesis [12][13][14] . These reports suggest that RvD1, RvD2, MaR1 and RvE1 are involved in the pathogenesis of RA. However, few reports have examined the role of other SPMs in immune cells and their involvement in in ammatory diseases.
Here we performed LM pro ling on in amed arthritic paws in a mouse model of RA, and found that RvD5 levels were elevated and correlated with arthritis disease activity. We demonstrated that RvD5 suppressed Th17 cells more strongly than RvD1. We also investigated the effects of RvD5 on the different cells involved in the pathology of RA in vitro and in vivo.

Results
RvD5 is elevated in the paws of arthritic SKG mice.
We injected SKG mice with Zymosan A (ZyA) (n = 8) and observed them for 16 weeks in comparison to mice not receiving ZyA (n = 5). In the arthritic SKG mice, joint in ammation was sustained for these 16 weeks (data not shown). At week 16, we euthanized the mice, collected their paws and used wide-targeted LC/MS/MS-based lipidomics analysis to investigate how LM pro les changed at the in ammatory sites in chronic arthritis (Fig. 1A, Table). In arthritic paws, arachidonic acid-derived pro-in ammatory mediators, such as PGE 2 , were signi cantly elevated relative to non-arthritic paws. Eicosapentaenoic acid (EPA)-or DHA-derived SPMs, such as RvE3, RvD3, RvD5, and MaR2, were also signi cantly increased in the arthritic paws. RvD1 showed a tendency to be increased (P = 0.062). Among these elevated SPMs, levels of RvD5 were most strongly correlated with arthritis disease activity (r = 0.892, P = 0.005) (Fig. 1B). These results suggested that RvD5 might be involved in the pathogenesis of arthritis.
Biosynthesis of AA -derived pro-in ammatory mediators might be increased in the arthritic paws.
We analyzed the ratio of LMs to precursors to identify whether elevated LMs were actively biosynthesized in arthritic paws. Compared to normal paws, the ratios of arachidonic acid (AA)-derived pro-in ammatory mediators to their precursors were signi cantly increased (PGF 2a , and TXB 2 ) or tended to be increased (PGE 2 , PGD 2 and 8iso-PGF 2a : corrected P-value: P > 0.05, uncorrected P-value: P < 0.05 with Mann-whitey U test with FDR-BH correction) in the arthritic paws (Fig. S1). On the contrary, ratio of AA-derived 15deoxy-Δ 12,14 -Prostaglandin J 2 to AA, which had been shown to ameliorate arthritis of collagen induced arthritis model 15 , tended to be decreased in the arthritic paws (corrected P-value: P > 0.05, uncorrected Pvalue: P < 0.05). There was no signi cant difference in EPA-derived and DHA-derived metabolites between arthritic paws and non-arthritic paws, except RvE1, RvE3 and RvD3 which were lower than the detectable range in the non-arthritic paws. These results suggested that the biosynthesis of AA-derived proin ammatory mediators might be increased in the arthritic paws.
We also analyzed ratios of pro-in ammatory LMs (PGs + TXB 2 + LTB 4 ) versus pro-resolving (15-deoxy-Δ 12,14 -Prostaglandin J 2 + Lipoxins + Rvs + PD1 + MaRs) LMs levels to clarify whether the imbalance of pro-in ammatory to pro-resolving LMs contributed to chronic arthritis. The ratios of pro-in ammatory LMs to pro-resolving LMs were signi cantly higher in arthritic paws than in non-arthritic paws (P < 0.01) (Fig. 1C). This result suggested that the insu ciently elevated SPMs and highly elevated proin ammatory LMs might be involved in the chronic persistent arthritis of SKG mice.

RvD1 and RvD5 suppress Th17 cell differentiation and facilitate regulatory T cell differentiation in vitro.
Different types of immune cells, such as helper T cells, dendritic cells (DCs), macrophages and OCs, are involved in the pathogenesis of joint in ammation in RA 4 . Therefore, we next examined whether DHAderived SPMs have any effects on these cells in vitro. First, we cultured CD4 + cells with SPMs under Th17-inducing conditions ( Fig. 2A). Consistent with a previous report, we found that RvD1 inhibited Th17 differentiation and facilitated the differentiation of Tregs 11 (Fig. 2B, C). Going beyond this, we also found that RvD5 inhibited the differentiation of Rorgt + Th17 cells and increased the population of Foxp3 + Tregs more potently than RvD1. Thus, while RvD1 caused a 33% reduction of Rorgt + Th17 cells, RvD5 reduced them by 66% at 500 nM. Similarly, RvD1 increased Foxp3 + Tregs by 26%, whereas RvD5 resulted in a 39% increase at 500 nM ( Fig. 2C). In contrast, RvD3 and MaR2 did not affect Th17 and Treg differentiation.
These results suggest that RvD5 suppresses the differentiation of Th17 cells and increases Tregs more effectively than RvD1.

RvD5 inhibits CD4 + T cell proliferation.
A previous report showed that RvD1 did not affect CD4 + T cell proliferation 11 . Therefore, we next examined the effect of RvD5 on CD4 + T cell proliferation and found that it was in fact suppressed in a dose-dependent manner (Fig. 3A, B). We con rmed that this was not seen for RvD1, and also showed that the same was true for RvD3 or MaR2, which had no effect on CD4 + T cell proliferation (data not shown).
RvD5 may not affect DC differentiation and activation, or GM-DM phenotypes in vitro.
We then examined the effect of RvD5 on myeloid cells. First, we stimulated bone marrow (BM) cells from SKG mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, with or without daily addition of RvD5 (Fig. S2A). We found that RvD5 did not suppress DC differentiation (Fig. S2B). To further explore the effect of RvD5 on DC activation, we incubated DCs with RvD5 before lipopolysaccharide (LPS) stimulation (Fig. S2C) and found that RvD5 had no inhibitory effects on CD86 + or MHC class + populations of CD11b + CD11c + cells (Fig. S2D).
To investigate the effect of RvD5 on macrophages, we cultured GM-DMs in an in ammatory environment 16 and stimulated them with LPS and IFNg to generate M1 phenotype cells. These were treated or not treated with RvD5 before LPS/IFNg stimulation (Fig. S2E). There was no effect on macrophage polarization (Fig. S2F). These results suggest that RvD5 may not have any effects on myeloid cells.
RvD5 decreases bone marrow-derived OC growth and interferes with osteoclastogenesis at the molecular level.
OCs play a pivotal role in the development of bone erosions in RA. We therefore investigated whether RvD5 has any effects on osteoclastogenesis. To this end, we cultured macrophage colony-stimulating factor (M-CSF)-induced bone marrow macrophages (BMMs) stimulated with receptor activator of NF-kB ligand (RANKL) to generate bone marrow-derived OCs, to which RvD5 was added daily (Fig. 4A). RvD5 treatment resulted in decreased formation of RANKL-induced TRAP + MNCs in a dose-dependent manner (Fig. 4B, C). We also studied cell viability under these conditions, and found that this was not affected by RvD5 (data not shown). Next, we examined the expression of OC-related genes. Levels of nuclear factor of activated T cells c1a (NFATc1a), known as a master transcription factor for OC differentiation, were decreased by RvD5 treatment in a dose-dependent manner (Fig. 4D). Many other genes associated with osteoclastogenesis, such as osteoclast-associated receptor (Oscar), acid phosphatase 5 (Acp5), cathepsin K (Ctsk), osteoclast stimulatory transmembrane protein (Ocstamp), dendritic cell-speci c transmembrane protein (Dcstamp), were also suppressed by RvD5 treatment. These results suggest that RvD5 may be involved in the pathogenesis of RA by inhibiting the differentiation of OCs.
RvD5 treatment tends to ameliorate arthritis in vivo.
Because RvD5 suppressed Th17 cell differentiation and facilitated Treg differentiation, inhibited CD4 + T cell proliferation and interfered with osteoclastogenesis, we hypothesized that RvD5 treatment might prevent arthritis progression in SKG mice. We administered RvD5 (1000 ng daily) or a physiological saline solution intraperitoneally to ZyA-induced SKG mice. RvD5 treatment tended to prevent and ameliorate arthritis, although these bene cial effects did not achieve statistical signi cance (Fig. S3).

Discussion
In the present study, we demonstrated that both pro-in ammatory mediators and SPMs are elevated in the arthritic paws in SKG mice, a model of chronic arthritis. Two recent studies have reported LC/MS/MSbased LM metabolomics analysis on arthritic paws in other model of arthritis 7,8 . Both studies used a K/BxN serum transfer model, which exhibits self-limited arthritis peaking at around 10 days. Levels of many pro-in ammatory mediators tended to increase during the in ammatory phase (days 4-8), and decrease again in the resolution phase (day 16). In contrast, levels of several SPMs including D-series resolvins were decreased during the in ammatory phase and peaked in the resolution phase. This study is the rst to perform LC/MS/MS-based LM metabolomics analysis in a chronic persistent arthritis model and we found that, in the chronic course, levels of many pro-in ammatory mediators remained very high and the ratios of pro-in ammatory to pro-resolving LMs levels were signi cantly higher in arthritic paws than in non-arthritic paws. Our results are consistent with a recent study showing that serum MaR1 levels in active RA patients are signi cantly lower than in inactive RA patients 10 . These ndings suggest that persistent high levels of pro-in ammatory mediators and insu ciently elevated SPMs contribute to the pathology of chronic joint in ammation.
Jonasdottir et al. 17 investigated lipid mediators in synovial uid from osteoarthritis and RA patients by LC/MS/MS analysis. The authors showed that AA, EPA, DHA and these metabolites were detectable in RA, and concentration ratios of several AA-derived metabolites to their precursors were higher in RA than in osteoarthritis. The results in human RA study are consistent with our ndings showing that concentration ratios of AA-derived pro-in ammatory mediators/AA were increased in mouse model of RA. These results suggest that pro-in ammatory mediators may be actively biosynthesized in the arthritic joints.
RvD5 was initially identi ed in media from ionophore-stimulated trout brain cells 18 . Previous studies have demonstrated that RvD5 is produced by M2 macrophages and has an enhancing effect on their phagocytosis 19,20 . A previous report showed that RvD5 is present in synovial uid from patients with RA 9 , but its role in the pathogenesis of this disease is unknown. To the best of our knowledge for the rst time, we here report that RvD5 suppressed Th17 cell differentiation and facilitated Treg differentiation. Higher percentages of circulating Th17 cells and higher concentrations of IL-17A have been reported in sera from RA patients 21,22 . A Th17/Treg imbalance has been suggested to be involved in the pathogenesis of RA 21 . Our ndings imply that increasing RvD5 levels might be able to correct Th17/Treg cell imbalances and suppress joint in ammation in RA.
RvD1 has been reported to inhibit bone resorption in a murine model of collagen antibody-induced arthritis 14 . A previous study showed that DHA exerted an inhibitory effect on OC formation which was counteracted by 5-lipoxygenase inhibition 23 . In the present study we demonstrated that RvD5 inhibits OC differentiation. Because 5-lipoxygenase acts as an essential enzyme to convert DHA to D-series resolvins, RvD5, in addition to other D-series resolvins, may suppress bone destruction in RA by suppressing OC differentiation.
In vivo RvD5 treatment tended to ameliorate arthritis and result in a low incidence of arthritis, although this effect did not achieve statistical signi cance. There are several possible reasons why this was the case. PGE 2 has been reported to promote Th17 cell differentiation and OC maturation 24 . LTB 4 induces IL-1 and TNF production by RA broblast-like synoviocytes and promotes neutrophil, macrophage and CD4 + T cell recruitment into the joint 25 . Because we measured extremely high levels of PGE 2 and LTB 4 in the in ammatory joints in SKG mice, high levels of pro-in ammatory SPMs might be the reason for the lack of a signi cant effect of RvD5 monotherapy in vivo. In addition, weak effects of RvD5 on DCs and macrophages might contribute to the result. Previous study demonstrated that RvD5 down-regulated genes of in ammatory markers in macrophages 19 . As for the effect of RvD5 on myeloid cells in this study, we might have underestimated its effect because serum (1% FBS) in the medium might affect the effects of SPMs on target cells. Also, standard deviation of in vivo experiment was much higher than we expected.
In conclusion, our results suggest that elevation of RvD5 is not simply a result of in ammation, but is involved in the pathogenesis of RA. Targeting the resolution of in ammation seems promising as a novel treatment of RA.

Conclusions
SPMs including RvD5 were found to be elevated in localized in ammation in animals with chronic arthritis. RvD5 suppressed Th17 cell differentiation and CD4 + T cell proliferation, facilitated Treg differentiation, and suppressed osteoclastogenesis. Further investigations are required to determine the effect of SPM on chronic arthritis and to determine cell targets in RA pathogenesis.

Ethical provisions.
This study was approved by the President of Kobe University after the review by Institutional Animal Care and Use Committee (Permission number: P 170703) and carried out according to the Kobe University Animal Experimentation Regulations. This study was carried out in compliance with the ARRIVE guidelines.

Animals.
SKG mice were purchased from CLEA Japan, Inc. They were housed in the Kobe University animal facility. All procedures were carried out in accordance with the recommendations of the Institutional Animal Care Committee of Kobe University.
Arthritis was induced in 8-10 weeks-old SKG mice by the injection of 2 mg/body ZyA intraperitoneally, as previously described 26 . The development and severity of arthritis was monitored using a previously described system for scoring clinical arthritis 27  CD4 + T cell isolation. SKG mice (3-4 weeks old) were sacri ced and splenocytes isolated after erythrocyte lysis using ACK Lysing Buffer. To isolate CD4 + T cells from single cell suspensions prepared from the splenocytes, we used a biotinylated mAb against CD4, streptavidin-coated magnetic beads and a manual MACS system (all from Miltenyi Biotec) according to the manufacturer's protocol.
T cell proliferation.
CD4 + T cells were incubated with 10 mM CFSE according to the manufacturer's protocol and were cultured in RPMI supplemented with 1% FBS, 1% P/St and 50 µM 2-ME for 3 days. RvD5 (1, 10, or 100 nM) was added daily from day 0. On day 3, cells were analyzed by ow cytometry.
Dendritic cell differentiation and activation.
BM cells (1.0 × 10 6 /well) were cultured for 3 days in RPMI 1640 containing GM-CSF (10 ng/ml). On day 4, non-adherent cells were collected and reseeded into 12-well plates at 2.0 × 10 6 /well with GM-CSF. On day 7, at which time we considered these cells to be GM-DMs, the medium was changed, and cells were stimulated with LPS (100 ng/ml) and IFN-g (50 ng/ml) for M1 polarization. On day 8, adherent cells were collected and cultured in 48-well plates in RPMI 1640 supplemented with 1% FBS and RvD5 (1, 10, 100, or 1000 nM). After 6 hours of RvD5 treatment, cells were evaluated by ow cytometry.
For surface staining, cells were washed with cell-staining buffer and incubated with anti-CD4, anti-CD11b, anti-CD11c, anti-F4/80, anti-CD86, anti-MHC class and anti-CD206 antibodies for 30 minutes at 4℃. For intracellular staining CD4 + T cells were treated with the nuclear factor xation and permeabilization buffer and stained with anti-Rorgt and anti-Foxp3 antibodies, according to the manufacturer's protocol. All samples were measured on a FACS Verse (BD Bioscience) and analyzed with FlowJo software (Tree Star).
Mouse BM cells were cultured on Petri dishes. After 3 hours, non-adherent cells were collected and seeded into 48-well plates at 5.0 × 10 4 /well or into 96-well plates at 2.0 × 10 4 /well in MEMa supplemented with 10% FBS, 1% P/St, and M-CSF (25 ng/ml). After preculture for 2 days, BMs were stimulated with M-CSF and RANKL (50 ng/ml). After a further 3 days, RvD5 (10, 100, or 500 nM) was added daily and cells were collected on day 5. Tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNCs; ≥ 3 nuclei) were visualized using a TRAP staining kit (Cosmo Bio Co., Ltd.) according to the manufacturer's protocol, and OCs were counted under a microscope (Keyence).
Total RNA was extracted using RNeasy Mini kits (Qiagen) and complementary DNA was synthesized by a QuantiTect Reverse Transcription Kit (Qiagen). Quantitative RT-PCR was performed using a QuantiTect SYBR Green PCR kit (Qiagen) with PikoReal system (Thermo Fisher Scienti c). GAPDH mRNA was used for internal normalization.

Statistical analysis.
Results were expressed as means and SEMs. Statistical analysis of LM levels was performed by Mann-Whitney U test with false-discovery rate (FDR)-BH correction. Correlations between disease activity and RvD5 levels were estimated by the Spearman's rank correction coe cient procedure. For comparisons of three or more groups, one-way ANOVA and Tukey's multiple comparison test were performed using GraphPad Prism 5 (GraphPad Software). P < 0.05 was considered statistically signi cant.