Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Abstract

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.

FIGURE 1.

A, the structure of xanthone and isocoumarin is shown. B, the structure of SsnB derived from NMR spectroscopy is shown. C, the structure of SsnB is confirmed by x-ray crystallography.

FIGURE 2.

SsnB inhibits TLR ligand-induced cytokine expression in mouse macrophages. Macrophages were treated with SsnB (100 μm), LPS (50 ng/ml), Pam3CSK4 (500 ng/ml), Fsl-1 (100 ng/ml), poly(I:C) (10 μg/ml), or ODN1668 (2.5 μm) either alone or as indicated for 6 h, and the expression levels cytokines were measured by quantitative real-time PCR. Bars represent the mean ± S.E. For each treatment, n = 3. *, p < 0.01, Student's t test.

FIGURE 3.

Mouse macrophages were treated with SsnB and LPS as indicated for 16 h. Cytokine levels in the medium were measured by ELISA. Bars represent the mean± S.E. For each treatment, n = 5. *, p < 0.01, versus LPS, Student's t test.

FIGURE 4.

Mouse macrophages with or without pretreatment with SsnB for 30 min were further cultured with SsnB and LPS for 30 min in serum-free DMEM. Cell lysate was used for Western blot detection of phosphorylation of signaling proteins. Shown is one experiment of three independent experiments. Statistical analysis of Western blots is shown in supplemental Fig. 3.

FIGURE 5.

SsnB acts on the TLR4 signaling pathway intracellularly. A, a diagram of the chimeric receptor is shown. The CD4-TLR4 chimeric receptor is composed of the extracellular mouse CD4 (mCD4) Ig domain and the transmembrane region and intracellular TIR domain of human TLR4 (hTLR4). PM, plasma membrane. B, THP-1 cells were transfected with CD4/TLR4 and pNFκB-Luc or control pCMV-Luc. 24 h after transfection the cells were treated with 0, 10, or 100 μm SsnB for 6 h. The cell culture medium was collected for secreted luciferase (relative luciferase units (RLU)) report assay. Bars represent the mean ± S.E. For each treatment, n = 6. *, p < 0.01 compared with 0 SsnB; #, p < 0.01 compared with 10 μm SsnB.

FIGURE 6.

SsnB disrupts TLR-MyD88 interaction. A, HEK293 cells were transiently co-transfected with FLAG-TLR4 or FLAG-TLR9 and pcDNA-MyD88-CFP. 24 h after transfection the medium was changed to DMEM with 1% FBS with or without indicated LPS (100 ng/ml), ODN1668 (2 μm), or SsnB (100 μm). After further incubation of 30 min, the cells were lysed. The cell lysates were used for Western blot detection of TLR4 or TLR9 and MyD88-CFP or used for co-immunoprecipitation (IP) by anti-TLR4 or anti-TLR9 antibody. The precipitates (P) and supernatants (S) were examined against anti-GFP antibody for detecting MyD88-CFP. Mouse Ig was used as a control for immunoprecipitation. Shown is the representative of three independent experiments. B, THP-1 cells (5 × 10⁵ cells/well in a 6-well plate) were transiently co-transfected with FLAG-TLR4 or FLAG-TLR2 or FLAG-TLR9 and pcDNA-MyD88-CFP using Lipofectamine^TM LTX reagent (1 μg total DNA for each well). 48 h after the transfection the medium was changed to fresh medium with 1% FBS containing the indicated TLR ligand and SsnB. After further incubation of 30 min, the cells were lysed. The cell lysates were used for Western blot detection of TLR4, TLR2, or TLR9 and MyD88-CFP or used for co-immunoprecipitation by anti-TLR4, anti-TLR2, or anti-TLR9 antibody. The precipitates (P) and supernatants (S) were examined against an anti-MyD88 antibody for detecting MyD88-CFP. Shown is the representative of two independent experiments TF, transfer.

FIGURE 7.

A, HEK293T cells at 80% confluence in 10-cm dish were co-transfected with FLAG-TLR4, pcDNA-MyD88-CFP, and NFkB-Luc plasmids (5 μg each). 16 h after the transfection the cells were split in 24-well plates at 0. 25 × 10⁶/well. The culture medium was changed to DMEM with 1% FBS with 100 ng/ml LPS and indicated concentration of SsnB. The culture medium was collected 16 h later for luciferase report assay. Luciferase activity readings of the samples were shown in the left panel. Half-maximal inhibitory concentration (IC₅₀) was shown in the right panel. Data represent the mean± S.E. For each treatment, n = 5. B, THP-1 cells were transiently transfected with NFkB-Luc plasmids (5 μg each 10-cm dish). 24 h after the transfection, the cells were split in 24-well plates at 0.1 × 10⁶/well. The culture medium was changed to RPMI1640 with 1% FBS with 100 ng/ml LPS and indicated concentration of SsnB. The culture medium was collected 24 h later for luciferase report assay. Luciferase activity (relative luciferase units (RLU)) readings of the samples were shown in the left panel. Estimated IC₅₀ was shown in the right panel. Data represent the mean ± S.E. For each treatment, n = 4.

FIGURE 8.

SsnB inhibits cytokine expression in the spleens of mice challenged by LPS. C57Bl/6 mice were injected intraperitoneally SsnB (100 μg) or the same volume vehicle 1 h before the injection of LPS (100 μg) or vehicle. Eight hours after LPS injection, the mice were sacrificed, and the spleens were homogenized for total RNA extraction and cDNA preparation. Quantitative real-time PCR was used to measure the cytokine expression levels. Each group contains three mice. Bars represent the mean ± S.E.