Pharmacologic repression of retinoic acid receptor-related orphan nuclear receptor γ is therapeutic in the collagen-induced arthritis experimental model

Abstract

Objective: The nuclear receptor retinoic acid receptor-related orphan nuclear receptor γ (RORγ; T cell-specific isoform RORγt) is a key regulator of Th17 cell differentiation, controlling the production of the inflammatory cytokine interleukin-17 (IL-17). Lipopolysaccharide (LPS) stimulation of monocytes leads to the induction of RORγ. We previously showed that the potent and selective inverse agonist of RORγ, SR2211, was effective at suppressing IL-17 production in EL4 cells. The aim of this study was to examine the effects of SR2211 treatment on proinflammatory cytokine expression in LPS-stimulated RAW 264.7 cells as well as on joint inflammation in vivo in mice with collagen-induced arthritis (CIA).

Methods: Collagen was injected into the tail of DBA mice, followed by a booster inoculation 21 days later. Three days prior to the booster inoculation, SR2211 was administered twice daily for 15 days. Thymus, spleen, and draining lymph nodes (DLNs) were then harvested, and Th17 cell differentiation and DLN stimulation were performed.

Results: Treatment of Th17 cells with SR2211 suppressed the expression and production of inflammatory cytokines. Likewise, SR2211 reduced inflammatory cytokine production in LPS-stimulated RAW 264.7 cells. Mice with CIA that received SR2211 twice daily for 15 days exhibited a statistically significant reduction in joint inflammation as compared to mice that received only vehicle. Interestingly, systemic Th1 cell activation was detected in SR2211-treated mice with CIA, as indicated by an increase in interferon-γ levels.

Conclusion: The findings of this study support the idea of targeting RORγ to therapeutically repress inflammatory T cell function and macrophage activation in humans with rheumatoid arthritis. Compounds such as SR2211 have potential utility for the treatment of inflammatory disease.

Fig. 1. The effect of SR2211 on IL17 gene expression and T_H17 cell differentiation

A) EL4 cells were pretreated with DMSO or SR2211 (0.1 or 1μM) for 20h followed by stimulation with PMA and ionomycin for 5 h. IL-17 and IL23R mRNA expression was quantitated and normalized to GAPDH. B) IFNγ or IL17 expression in splenocytes cultured under T_H1 polarizing conditions or T_H17 polarizing conditions with vehicle control (DMSO) or SR2211 (0.1μM or 1μM) for 3 days. Graphs represent the average concentration of IFNγ or IL17 cytokines (n=3). All error bars denote SEM. **p < 0.01, ***p < 0.001 by unpaired t-test.

Fig. 2. The Induction of RORγ in LPS stimulated RAW264.7 cells and the inhibition of pro-inflammatory cytokines by SR2211

A) RAW264.7 cells were incubated with 1μg/mL LPS for 6h and measured the mRNA of mouse RORγ. B) The histogram represented the expression of intracellular protein of RORγ in RAW264.7 cells (n=3). Graph represent average of MFI (median fluorescence intensity). C) IL1β, IL6, and TNFα mRNA levels are measured by pre-incubation with 5μM SR2211 for 18 hours then added LPS (1μg/mL) with 5μM SR2211 for 6h. All error bars denote SEM. *p < 0.05, ***p < 0.001 by unpaired t-test.

Fig 3. The therapeutic effect of SR2211 on arthritis onset

A) Treatment with SR2211 (20mg/kg) or dexamethasone (0.25mg/kg) suppressed the clinical severity of CIA [Vehicle (circle, n=13) or SR2211 (closed circle, n=7) or dexamethasone (red square, n=15)]. Clinical score was monitored every day after boosting during two weeks. B) Efficacy of SR2211 was dose-responsive, and C) Photograph inserts of a paw from one vehicle-only treated mouse and one from a mouse treated at 20mg/kg SR2211. Haematoxylin & eosion staining on paraffin sections of the joints on day 32 post immunization showing bone erosion (arrows) and infiltrated immune cells. The % infiltrated cells were counted on identical regions of swollen figures with vehicle group set to 100%. n= 3~4 mice/ group. All error bars denote SEM. *p < 0.05, ***p < 0.001 by unpaired t-test.

Fig. 4. Stimulation of systemic T_H1 cells by SR2211

Draining lymph nodes from Vehicle group or SR2211 treating group was isolated and activated with α-CD3 and α-CD28 mAb for 24h. IFNγ and IL-17 were measured by intracellular staining. Graphs represent the average percentage of IFNγ and IL17 expressing cells normalized to vehicle control (n=6). Error bars denote SEM. ***p < 0.001 by unpaired t-test.