Effect of resolvin D5 on T cell differentiation and osteoclastogenesis analyzed by lipid mediator profiling in the experimental arthritis

Abstract

Resolvins, are specialized pro-resolving mediators (SPMs) derived from n-3 polyunsaturated fatty acids. They contribute actively to the resolution of inflammation, but little is known concerning their role in chronic inflammation, such as in rheumatoid arthritis (RA). Here, we performed lipid mediator (LM) profiling in tissues from the paws of SKG arthritic mice using lipid chromatography (LC)/mass spectrometry (MS)/MS-based LM metabololipidomics. We found elevated levels of SPMs including resolvin D5 (RvD5) in these tissues. Moreover, RvD5 levels were significantly correlated with arthritis disease activity. From experiments to assess the role of RvD5 in the pathology of RA, we concluded that RvD5 suppressed Th17 cell differentiation and facilitated regulatory T cell differentiation, as well as inhibiting CD4⁺ T cell proliferation. Furthermore, RvD5 attenuated osteoclast differentiation and interfered with osteoclastogenesis. Targeting the resolution of inflammation could be promising as a novel treatment for RA.

Figure 1

RvD5 is elevated in arthritis and correlates with the degree of arthritis. (A) SKG mice were injected with ZyA (2 mg/body i.p.) on day 0. On day 112, paws were removed and analyzed. (B) LM quantified using LC/MS/MS-based LM profiling (control group, n = 5, arthritis group, n = 8). Bars represent mean ± SEM. *P < 0.05, by Mann–Whitney U test and FDR-BH correction. (C) Correlation between disease activity and RvD5 level (Spearman’s rank correlation coefficient). (D) Ratios of pro-inflammatory versus pro-resolving lipid mediator levels. Bars represent mean ± SEM. **P < 0.01, by Mann–Whitney U test.

Figure 2

RvD5 suppresses Th17 cell differentiation and facilitates Treg differentiation. (A) Schematic representation of the Th17 cell culture conditions and experiment protocol. CD4⁺ T cells were isolated from 3–4 week-old SKG mice and cultured for 5 days in wells pre-coated with anti-CD3 and anti-CD28, in the presence of TGF-β, IL-6, anti-IFNγ antibody, anti-IL-4 antibody, and with or without daily addition of RvD5 (1–100 nM). On day 5, cells were stained with anti-CD4, anti-Rorγt and anti-Foxp3 antibodies. (B) Frequencies of Th17 cells (Rorγt⁺/ CD4⁺) and Tregs (Foxp3⁺/ CD4⁺) estimated by flow cytometry. Data are representative of five independent experiments. (C) Frequencies of Th17 cells and Tregs in each group, by flow cytometry. (D) CD4⁺ T cells were cultured under Th17-inducing conditions for 3 days with or without daily addition of RvD5 (500 nM) and then stimulated with PMA/Ionomycin for 6 h. Cells were stained with anti-CD4, anti-IL-17A antibodies. Frequencies of IL-17A⁺/CD4⁺ cells estimated by flow cytometry. Data are representative of seven independent experiments. (E) Frequencies of IL-17A⁺/CD4⁺ cells in each group, by flow cytometry. Bars represent mean ± SEM. **P < 0.01, *P < 0.05, by one-way analysis of variance and Tukey’s multiple comparison test.

Figure 3

RvD5 suppresses CD4⁺ T cell proliferation. (A) CD4⁺ T cell proliferation measured by CFSE fluorescence. Data are representative of five independent experiments. (B) Frequencies of proliferating T cells in each group, analyzed by flow cytometry. Bars represent mean ± SEM. **P < 0.01, *P < 0.05, by one-way analysis of variance and Tukey’s multiple comparison test.

Figure 4

RvD5 suppresses RANKL-induced osteoclastogenesis. (A) Schematic representation of RANKL-induced osteoclastogenesis. BM cells were isolated from 8–10-week-old SKG mice and cultured for 2 days with M-CSF. BMMs were then incubated with M-CSF and RANKL, with or without daily RvD5 (10–500 nM). Cells were fixed and stained for TRAP after 5 days of culture. (B) Representative images of TRAP staining. (C) TRAP-positive MNCs were counted. (D) Relative cell viability. (E) The mRNA expression levels were quantified by qPCR. Messenger RNA levels were normalized to GAPDH. Bars represent mean ± SEM. **P < 0.01, by one-way analysis of variance and Tukey’s multiple comparison test.