Identification of the synthetic cannabinoid R(+)WIN55,212-2 as a novel regulator of IFN regulatory factor 3 activation and IFN-beta expression: relevance to therapeutic effects in models of multiple sclerosis

Abstract

β-Interferons (IFN-βs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.

FIGURE 1.

R(+)WIN55,212-2 negatively regulates TLR3/4-induced activation of NF-κB and expression of TNF-α. HEK293-TLR4 (A and B) and HEK293-TLR3 (C and D) cells were cotransfected with plasmids encoding NF-κB-regulated firefly luciferase (80 ng) and constitutively expressed TK Renilla luciferase (20 ng). 24 h post-transfection cells were treated in the absence or presence of R(+)WIN55,212-2 (5–50 μm) (A) and S(−)WIN55,212-2 (B) (5–50 μm) for 1 h prior to treatment with LPS (100 ng/ml) (A and B) and poly(I·C) (25 μg/ml) (C and D) for 6 h. Cell lysates were assayed for firefly luciferase activity and normalized for transfection efficiency using Renilla luciferase activity. Data are presented relative to vehicle-treated cells and represent the mean ± S.E. of triplicate determinations from three independent experiments. E–H, primary mouse astrocytes were seeded into 12-well plates, pre-treated with R(+)WIN55,212-2 (5–50 μm) (E and G) or S(−)WIN55,212-2 (5–50 μm) (F and H) for 1 h and stimulated with (E and F) LPS (100 ng/ml) or (G and H) poly(I·C) (25 μg/ml) for 6 h. Supernatants were analyzed for TNF-α production using sandwich ELISA. Data are presented as the mean ± S.E. of triplicate determinations from six animals and are representative of two independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with vehicle-treated cells. +++, p < 0.001 compared with LPS- or poly(I·C)-treated cells.

FIGURE 2.

R(+)WIN55,212-2 augments TLR3-induced activation of IRF3 and expression of IFN-β. HEK293-TLR4 (A) and HEK293-TLR3 (B and C) cells were cotransfected with pFA-IRF3 (30 ng) and pFR-regulated firefly luciferase (60 ng) and constitutively expressed TK Renilla luciferase (20 ng). Transfected cells were left overnight and then cells were treated in the absence or presence of R(+)WIN55,212-2 (5–50 μm) (A and B) and S(−)WIN55,212-2 (5–50 μm) (C) for 1 h prior to treatment with/without LPS (100 ng/ml) (A) or poly(I·C) (25 μg/ml) (B and D) for 6 h. D, HEK293-TLR4, and HEK293-TLR3 (E and F) cells were cotransfected with pFA-IRF7 (25 ng) and pFR-regulated firefly luciferase (60 ng), left overnight, and treated in the absence or presence of R(+)WIN55,212-2 (5–50 μm) (D and E) and S(−)WIN55,212-2 (5–50 μm) (F) for 1 h prior to treatment with LPS (100 ng/ml) (D) or poly(I·C) (25 μg/ml) (E and F) for 6 h. HEK293-TLR3 (G and I) and U373-CD14 (H) cells were cotransfected with IFN-β promoter (G and H) or positive regulatory domains I-III-regulated firefly luciferase (80 ng) (I) and constitutively expressed TK Renilla luciferase (20 ng), left overnight, and treated in the absence or presence of R(+)WIN55,212-2 (1–50 μm) for 1 h prior to treatment with poly(I·C) (25 μg/ml) for 6 h. In all cases (A–I) cell lysates were assayed for firefly luciferase activity and normalized for transfection efficiency using Renilla luciferase activity and represent the mean ± S.E. of triplicate determinations from three independent experiments. J and K, BMDMs were treated in the absence or presence of R(+)WIN55,212-2 (5–50 μm) for 1 h prior to treatment with LPS (100 ng/ml) (J) or poly(I·C) (25 μg/ml) (K) for 18 h. cDNA was generated and assayed by quantitative real time PCR for levels of Ifn-β mRNA. The expression level of Ifn-β was normalized relative to expression of the housekeeping gene Gapdh and represent the mean ± S.E. of triplicate determinations from three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with vehicle-treated cells. +, p < 0.05; ++, p < 0.01; and +++, p < 0.001 compared with LPS- or poly(I·C)-treated cells.

FIGURE 3.

R(+)WIN55,212-2 regulates TLR3 signaling in a cannabinoid receptor-independent manner. A, total cellular RNA was prepared from HEK293 cells and subjected to first strand cDNA synthesis using SuperScript II reverse transcriptase and random oligonucleotide primers. PCR amplification was performed using Taq DNA polymerase and primers to selectively amplify regions of CB₁, CB₂, and GAPDH cDNA. B, D, and F, HEK293-TLR3 cells were cotransfected with pFA-IRF3 (30 ng) and pFR-regulated firefly luciferase (60 ng) and constitutively expressed TK Renilla luciferase (20 ng). Transfected cells were left overnight and then cells were pre-treated (1 h) with the inhibitors SR141716 (1 μm) (B), SR144528 (1 μm) (D), and PTX (50 ng/ml) (F) prior to exposure to R(+)WIN55,212-2 (20 μm; 1 h), and then stimulated with poly(I·C) (25 μg/ml) for 6 h. Cell lysates were assayed for firefly luciferase activity and normalized for transfection efficiency using Renilla luciferase activity. C, E, and G, HEK293-TLR3 cells were pre-treated (1 h) with the inhibitors SR141716 (1 μm) (C), SR144528 (1 μm) (E), and PTX (50 ng/ml) (G) prior to exposure to R(+)WIN55,212-2 (20 μm; 1 h), and then stimulated with poly(I·C) (25 μg/ml) for 4 h. cDNA was generated and assayed by quantitative real time PCR for levels of IFN-β mRNA. The expression level of IFN-β was normalized relative to expression of the housekeeping gene GAPDH. H, HEK293 cells were pre-treated with or without PTX (100 ng/ml; 24 h), SR141716 (SR1; 1 μm for 1 h), and SR144528 (SR2; 1 μm for 1 h) prior to treatment with ACEA (100 nm for 1 h) or JWH133 (100 nm for 1 h). Cells were then incubated with 3-isobutyl-1-methylxanthine (500 μm for 15 min) and stimulated with forskolin (30 μm for 30 min). Lysates were harvested and assessed for levels of intracellular cAMP using a cAMP parameter kit. Data represent the mean ± S.E. of triplicate determinations from three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with vehicle-treated cells. +, p < 0.05; and ++, p < 0.01 compared with poly(I·C)-treated cells (B-G) and forskolin-treated cells (H). $$, p < 0.01 compared with cells treated with ACEA/JWH133 in the presence of forskolin.

FIGURE 4.

R(+)WIN55,212-2 augments the TLR3/TRIF/TBK1 signaling axis and promotes nuclear localization of IRF3. A, HEK293-TLR3 cells were cotransfected with pFA-IRF3 (30 ng), pFR-regulated firefly luciferase (60 ng), TRIF reporter constructs (50 ng), and constitutively expressed TK Renilla luciferase (20 ng). Transfected cells were left overnight and treated in the absence or presence of R(+)WIN55,212-2 (5–50 μm) for 6 h. Cell lysates were assayed for firefly luciferase activity and normalized for transfection efficiency using Renilla luciferase activity. Data are presented relative to vehicle-treated cells and represent the mean ± S.E. of triplicate determinations from three independent experiments. B, TRIF-deficient BMDMs were pre-treated (1 h) with R(+)WIN55,212-2 (20 μm) and then stimulated with poly(I·C) (25 μg/ml) for 18 h. cDNA was generated and assayed by quantitative real time PCR for levels of Ifn-β mRNA. The expression level of Ifn-β was normalized relative to expression of the housekeeping gene Gapdh and represents the mean ± S.E. of triplicate determinations from three independent experiments. C and D, primary mouse astrocytes were seeded into 6-well plates and treated with poly(I·C) (25 μg/ml) (C) for various time points (5–360 min) or pre-treated with R(+)WIN55,212-2 (20 μm; 1 h) (D) prior to stimulation with poly(I·C) (25 μg/ml) for 1 h. Cell lysates were subsequently subjected to Western immunoblotting using anti-phospho-Ser³⁹⁶ IRF3, anti-total IRF3, and anti-β-actin antibodies (lower panels). All immunoblots were subjected to densitometric analysis with levels of phospho-IRF3 normalized to total levels of IRF3 (upper panels). Densitometic data are representative of 8 (C) and 6 (D) independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus non-transfected (A) and vehicle-treated cells (C and D). +, p < 0.05; and +++, p < 0.001 compared with vehicle treated TRIF-transfected cells. E, primary mouse astrocytes were grown in chamber slides and pre-treated (1 h) with R(+)WIN55,212-2 (20 μm) or S(−)WIN55,212-2 (20 μm) for 1 h prior to poly(I·C) (25 μg/ml) exposure for 1 h. Cells were fixed, mounted in anti-fade medium with DAPI, and visualized using confocal microscopy. Confocal images were captured using a UV Zeiss 510 Meta System laser scanning microscope equipped with the appropriate filter sets. Data analysis was performed using the LSM 5 browser imaging software. Images are representative of three independent experiments. Scale bars are 20 μm. F, primary astrocytes were pre-treated with or without R(+)WIN55,212-2 (20 μm) for 1 h prior to stimulation in the absence or presence of poly(I·C) (25 μg/ml; 1 h). Cytosolic and nuclear fractions were prepared and subsequently subjected to Western immunoblotting using anti-total IRF3 and anti-β-actin antibodies. Blots are representative of data obtained from 6 animals.

FIGURE 5.

Protective effects of R(+)WIN55,212-2 in EAE are mediated by IFN-β. A, PLP-immunized mice develop clinical symptoms of EAE from day 5 post-immunization, with disease severity peaking on day 16 followed by a relapse on day 26. Mice treated with R(+)WIN55,212-2 (administered (20 mg/kg) intraperitoneally on days 0, 1, 2, 3, 4, and 5 after immunization) showed delayed development of EAE and attenuated disease severity. PLP-immunized mice treated with R(+)WIN55,212-2 and an anti-IFN-β antibody (administered intraperitoneally (2 × 10³ neutralizing units) on days 3 and 5 after PLP immunization) were not protected. B, representative images of Luxol fast blue-stained spinal cord sections from untreated mice, PLP-treated, PLP + WIN-treated, and PLP + WIN + αIFNβ-treated mice illustrating the extent of demyelination and lymphocytic inflammation. The posterior funiculi of the spinal cord were observed under high power (right panels). Images are representative of data from 4 to 8 animals per treatment group. Scale bars are 200 and 50 μm. Spinal cords were sectioned and stained with hematoxylin and eosin and quantified for spinal cord inflammation (C) and extent of demyelination (D) using Luxol fast blue-stained spinal cord sections in treated groups. cDNA was generated from spinal cords and assayed by quantitative real time PCR for relative levels of Gfap mRNA (E) and Cd11b mRNA (F) from vehicle-treated, PLP-treated, PLP + WIN-treated, and PLP + WIN + αIFNβ-treated mice. The expression level of Gfap and Cd11b was normalized relative to expression of the housekeeping gene Gapdh and represent the mean ± S.E. of triplicate determinations from 4 to 8 animals per treatment group. Cytosolic fractions were prepared from the spinal cord of vehicle-treated, PLP-treated, PLP + WIN-treated, and PLP + WIN + αIFNβ-treated mice. Cell lysates were subsequently subjected to Western immunoblotting using anti-phospho IκBα, anti-total IκBα, and anti-β-actin antibodies. Blots are representative of data from 4 to 8 animals per treatment group. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 for differences between WIN-treated mice and other groups.

FIGURE 6.

R(+)WIN55,212-2 induces IFN-β expression in PBMCs from MS subjects. A–H, PBMCs prepared from healthy subjects (A, C, E, and G) and MS patients (B, D, F, and H) were seeded into 24-well plates, pre-treated with R(+)WIN55,212-2 or S(−)WIN55,212-2 (5–50 μm) for 1 h, and stimulated with poly(I·C) (25 μg/ml) for 3 h. A–D, cDNA was generated and assayed by quantitative real time PCR for relative levels of IFN-β mRNA. The expression level of IFN-β was normalized relative to expression of the housekeeping gene GAPDH and represent the mean ± S.E. of triplicate determinations from three patients. Supernatants were analyzed for TNF-α (E and F) and IL-8 (G and H) production using sandwich ELISA. Data are presented as the mean ± S.E. of triplicate determinations from three patients. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with vehicle-treated cells (A, C, E, F, G, and H) or cells treated with R(+)WIN55,212-2 in the presence of poly(I·C) (B). ++, p < 0.01, and +++, p < 0.001 compared with poly(I·C)-treated cells.