Resolvin D1 Improves the Treg/Th17 Imbalance in Systemic Lupus Erythematosus Through miR-30e-5p

Abstract

Resolvin D1 (RvD1) prompts inflammation resolution and regulates immune responses. We explored the effect of RvD1 on systemic lupus erythematosus (SLE) and investigated the correlation between RvD1 and Treg/Th17 imbalance, which is one of the major factors contributing to the pathogenesis of disease. SLE patients and healthy controls were recruited to determine plasma RvD1 levels. MRL/lpr lupus model was used to verify rescue of the disease phenotype along with Treg/Th17 ratio. Purified naive CD4+ T cells were used to study the effect of RvD1 on Treg/Th17 differentiation in vitro. Furthermore, small RNA Sequencing and transfection were performed successively to investigate downstream microRNAs. The result showed that the RvD1 level was significantly lower in active SLE patients compared with inactive status and controls. Moreover, The SLE disease activity index (SLEDAI) score had a significant negative correlation with RvD1 level. As expected, RvD1 treatment ameliorated disease phenotype and inflammatory response, improved the imbalanced Treg/Th17 in MRL/lpr mice. In addition, RvD1 increased Treg while reduced Th17 differentiation in vitro. Furthermore, miR-30e-5p was verified to modulate the Treg/Th17 differentiation from naïve CD4+ T cells as RvD1 downstream microRNA. In conclusion, RvD1 effectively ameliorates SLE progression through up-regulating Treg and down-regulating Th17 cells via miR-30e-5p.

Keywords: Resolvin D1; T helper 17 cells; miR-30e-5p; regulatory T cells; systemic lupus erythematosus.

Figure 1

Plasma RvD1 level in SLE patients. (A) The levels of RvD1 in plasma samples from healthy controls, patients with inactive and active SLE (n=8 per group). (B) The SLEDAI score had a significant negative correlation with RvD1 level (r=-0.721, p=0.002). All data are mean ± SEM. **p < 0.01.

Figure 2

RvD1 influence the Treg/Th17 balance in MRL/lpr mice. (A) Quantification with ELISA of RvD1 in the plasma of MRL/lpr mice at 4, 8 and 12 weeks of age. (B) RvD1 treatment scheme (5μg/kg) injections were performed every three days for 2 months, starting from 8 weeks of age. (C, D) Representative FACS analysis of Treg and Th17 of spleens and cervical lymph nodes. All data are mean ± SEM. n=5 in MRL/lpr group; n=6 in MRL/lpr+RvD1 treated group. *p < 0.05; **p < 0.01.

Figure 3

RvD1 treatment ameliorates disease phenotype and inflammatory response in MRL/lpr mice. RvD1 treatment reduced spleen index (A), lymph node size (B), plasma levels of total IgG (C), anti-dsDNA antibodies (D), ANA (E), proteinuria (F), glomerular enlargement and hypercellularity (G), pro-inflammatory cytokines (TNF-α, IFN-γ and IL-6) (H) and a Th17 cell-related cytokine (IL-17) (I), while increase Treg cell-related cytokines (IL-10 and TGF-β) (I) in MRL/lpr mice. All data are mean ± SEM. n=5 in MRL/lpr group; n=6 in MRL/lpr+RvD1 treated group. *p < 0.05; **p < 0.01.

Figure 4

RvD1 modulates the proliferation and differentiation of Treg and Th17 in PBMCs and naïve CD4+ T cells in SLE patients. (A) The percentages of Treg and Th17 in human PBMCs with and without RvD1 treatment (n=7). (B) Flow cytometric analysis of Treg and Th17, which were differentiated from human naïve CD4+ T cells in the presence or absence of RvD1 (n=5). (C) The level of TGF-β, IL-10, and IL-17 in the culture supernatant were detected by ELISA (n=5). All data are mean ± SEM. *p < 0.05.

Figure 5

Effect of miR-30e-5p on the differentiation of naive CD4+ T cells from patients with SLE. (A) Venn diagram and heatmap analysis of miRNA expression profiles in Treg and Th17 differentiation from naive CD4+ T cells before and after RvD1 treatment. (B) The expression of miR-30e-5p, miR-32-5p, miR-26a-2-3p and let-7a-3p were verified by qPCR in independent SLE patients (n=10). (C, D) Representative flow cytometric plots indicated the percentage of Treg and Th17 cells polarized from naive CD4+ T cells transfected with miR-30e-5p mimic or inhibitor. (E) The level of TGF-β1, IL-10, and IL-17 in supernatant were detected by ELISA. All data are mean ± SEM. n=5 per group. ***p < 0.001.