Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation

Abstract

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Fig. 1.

Agonist activity of oxysterols in a cell-based RORγ reporter assay and direct binding to RORγ LBD. (A) Effect of 27-OHCs in reversing the inhibitory effect of UA in a cell-based chimeric RORγ reporter assay and inhibition of binding of ³H-25-OHC to the RORγ ligand binding domain. Shown in Upper are dose titration curves from representative experiments. For the reporter assay, oxysterols (starting at 6 μM; 1:3 serial dilutions) were tested in duplicates in the presence (0% of response) or absence (100% of response) of 1 μM UA. For the ³H-25-OHC/RORγ LBD competition binding assay, oxysterols (starting at 30 μM; 1:3 serial dilutions) were also tested in duplicates. Percent inhibition values were calculated based on without RORγ LBD protein as 100% and DMSO only as 0%. EC₅₀ or IC₅₀ values were calculated using GraphPad Prism 5. K_i values were calculated based on K_i = IC₅₀/([³H-25-OHC]/K_d + 1). Average ± SD (n = or > 3) of EC₅₀ or K_i values and efficacy values at the highest tested concentration are shown in the table. (B) Effect of 7β/α, 27-OHC vs. 7α, 25-OHC on binding of coactivator NCOA3 and corepressor NCORNR peptides by RORγ LBD in a surface plasmon resonance binding assay.

Fig. 2.

7β, 27-OHC and 7α, 27-OHC but not 7α, 25-OHC promoted IL-17 production of mouse and human Th17 cells in vitro. Shown are data from representative flow cytometry intracellular staining analysis of IL-17A and IFN-γ in mouse or human CD4⁺ T cells. (A) Purified total CD4⁺ T cells from WT and RORγt KO mice were activated under Th17-polarizing condition for 3 d. (B) Purified human naïve CD4⁺ T cells (CD45RO⁻, CCR6⁻) activated under Th17-polarizing condition for 10 d. DMSO (vehicle) or 1 μM UA was added 2 h before cell stimulation. Oxysterols (6 μM for mouse T cells and 0.3 μM for human T cells) were added at the start of culture. Statistical analyses of data from several independent experiments are shown in SI Appendix, Fig. S4.

Fig. 3.

Defective production of 27-OHCs and IL-17–producing cells in Cyp27a1 KO mice. (A) Spleen levels of 27-OHCs and 7-OHCs of Cyp27a1 KO vs. WT mice. Oxysterols in spleen samples (n = 5) were measured using deuterated oxysterols as internal controls as described in SI Appendix. (B) Flow cytometry intracellular staining analysis for IL-17A and IFN-γ in spleen CD4⁺ and γδ⁺ T cells from Cyp27a1 KO vs. WT mice. Total splenocytes were activated under Th17-polarizing condition for 3 d. RT-PCR analyses were performed on selected IL-17 pathway genes from mRNA samples (n = 2–3). RQ, relative quantification. (C) Defective in vitro Th17 differentiation of naive CD4⁺ T cells from Cyp27a1 KO mice. Purified naive CD4⁺ T cells of Cyp27a1 KO and WT controls were activated under Th17-polarizing condition for 6 d. DMSO (vehicle) or 7β, 27-OHC (6 μM) was added at the beginning of the culture. (D) Defective in vivo Th17 differentiation in Cyp27a1 KO mice. Cyp27a1 KO and WT mice were immunized with ovalbumin/complete Freund’s adjuvant (OVA/CFA); 7 d later, draining lymph node (LN) cells were harvested and restimulated with OVA in vitro for 2 d. IL-17A, IFN-γ, and IL-22 production from cultures of draining LN cells were determined by flow cytometry intracellular staining analysis and ELISA. Statistics by two-tailed, unpaired Student t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Dosing of 7β, 27-OHC in mice enhanced IL-17 production in vivo. (A) Effect on LN IL-17⁺ CD4⁺ and γδ⁺ T cells. B6 mice (n = 3) were dosed with vehicle or 7β, 27-OHC [60 mg/kg s.c. bis in die (BID)] for 3 d. Inguinal lymph node (LN) cells were harvested and stimulated with phorbol myristate acetate/inomycin for 4 h before intracellular staining. Data in the bar graph are averages ± SEMs of three mice per group. (B) Effect on IL-17 production in the OVA/CFA priming model. Three groups of B6 WT mice (n = 3) were immunized with OVA/CFA and dosed with vehicle, UA, (150 mg/kg i.p. every other day), or UA + 7β, 27-OHC (60 mg/kg s.c. BID); 7 d later, draining LN cells were harvested and restimulated with OVA in vitro for 2 d. IL-17A and IFN-γ production from cultures of draining LN cells were determined by flow cytometry intracellular staining analysis and ELISA. Data in bar graph are averages ± SEMs. Statistics by two-tailed, unpaired Student t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

³H-7β/α-OHC were preferentially converted into ³H-7β/α, 27-OHC in mouse Th17 but minimally in Th1 T cells. (A) Conversion of ³H-7β-OHC or 7α-OHC by mouse Th17 and Th1 cells in culture (purified total CD4⁺ T cells activated under Th17 or Th1 conditions for 3 d). Conditioned media were fractionated using HPLC as described in SI Appendix. Radio activities of 50 μL 1-min fractions were determined. Data shown were from a representative experiment of three independent experiments. (B) Conversion of ³H-7β-OHC by cultured Th17 cells differentiated from WT and Cyp27a1 KO naïve CD4⁺ T cells. Conditioned media were purified as above. Fractions containing the converted products were pooled and further purified by another HPLC; 30-s fractions were collected to resolve converted species, which resulted in peaks A and B. MS analysis revealed that peak B was consistent with being 7β, 27-OHC. (C) HPLC fractionation of conditioned medium from ³H-7β-OHC–spiked culture of untransfected COS7 cells or COS7 cells transiently expressing human CYP27A1 protein. CPM, counts per minute.