Figure 1.
TLR6 restores PDG-mediated inhibition of trophoblast IL-6 expression.
(A) The human first trimester trophoblast cell line, 3A, which lacks TLR6 (TLR6-), was treated with no treatment (NT) or peptidoglycan (PDG) at 80μg/ml for 3-24h, after which total RNA was isolated and IL-6 mRNA measured by qRT-PCR using GAPDH as an internal control (FC = fold change). After 12h, PDG treatment significantly reduced IL-6 mRNA levels (n=7). (B) Trophoblast cells either lacking TLR6 (TLR6-) or stably transfected to express TLR6 (TLR6+) were treated with NT or PDG (80μg/ml). After 12h, IL-6 mRNA was measured by qRT-PCR. The presence of TLR6 significantly reversed PDG-induced inhibition of IL-6 mRNA expression (n=5). *p<0.05, **p<0.001 relative to the NT control unless otherwise indicated.
Figure 2.
Inhibition of trophoblast NFκB activity reduces IL-6 mRNA expression and induces apoptosis.
TLR6- trophoblast cells were treated with no treatment (NT) or a NF-κB inhibitor. (A) After 12h treatment with the NF-κB inhibitor, IL-6 mRNA levels were significantly reduced (n=5). (B) After 24h, the NF-κB inhibitor significantly elevated caspase-3 activity (n=4). *p<0.05; **p<0.001 relative to the NT control.
Figure 3.
TLR6 restores PDG-mediated inhibition of trophoblast NF-κB p65 expression.
TLR6- or TLR6+ trophoblast cells were treated with no treatment (NT) or PDG (80μg/ml) for (A & B) 12h after which RNA was extracted, or (C - F) 48h, after which protein was collected. mRNA levels of (A) NF-κB p65 (n=4) and (B) NFKB1 (n=5) were measured by qRT-PCR. PDG treatment significantly reduced NF-κB p65, but not NFKB1, mRNA levels in the TLR6- cells, and this was completely reversed by the presence of TLR6. (C) Protein levels of total NF-κB p65 and NFKB1 (p105 and p50) were analyzed by Western blot. A representative immunoblot is shown. Barcharts below show quantification of (D) p65, (E) p105, and (F) p50 protein expression as determined by densitometry with normalization to β-actin. PDG treatment significantly reduced NF-κB p65 (but not NFKB1 p105 or p50) protein levels in the TLR6- cells, and this was reversed by the presence of TLR6 (n=3-5). *p<0.05 relative to the NT control unless otherwise indicated.
Figure 4.
Regulation of miRs in PDG-treated trophoblast cells.
TLR6- or TLR6+ trophoblast cells were treated with no treatment (NT) or PDG (80μg/ml) for 12h, after which RNA was collected and the expression of: (A) miR-329; (B) miR-23a; (C) miR-let-7c; (D) miR-149; and (E) miR-23b was measured by qRT-PCR. Data are presented as fold change (FC) in miR expression after normalization to the endogenous control, miR-374. PDG treatment significantly upregulated trophoblast miR-329, miR-23a, miR-let-7c, and miR-23b expression in TLR6-, and this was significantly inhibited by the presence of TLR6 (TLR6+). *p<0.05 relative to the NT control unless otherwise indicated. Data are from 5-7 independent experiments.
Figure 5.
miR-329 regulates PDG-induced trophoblast apoptosis and inhibition of IL-6 expression by targeting NF-κB p65.
(A) TLR6- trophoblast cells were transfected with an anti-miR-inhibitor scramble sequence labeled with Cy3. After 24h, uptake efficiency was evaluated by fluorescent microscopy and a 99% transfection efficiency was observed. (B - D) TLR6- trophoblast cells were transfected with either an anti-miR scramble sequence or a specific anti-miR-329 inhibitor. Post-transfection, cells were treated with either NT or PDG (80μg/ml) for 12h, after which (B) NF-κB p65 mRNA and (C) IL-6 mRNA levels were measured by qRT-PCR. (D) After 48h caspase-3 activity was measured. Treatment of scramble control cells with PDG significantly reduced NF-κB p65 and IL-6 mRNA expression levels and upregulated caspase-3 activity, and these responses were significantly reversed by the presence of the anti-miR-329 inhibitor. *p<0.05 relative to the NT control unless otherwise indicated (n=3-5).
Figure 6.
miR-23a and miR-let-7c regulate PDG-mediated inhibition of IL-6 expression.
TLR6- trophoblast cells were transfected with an either: an anti-miR scramble sequence; a specific anti-miR-23a inhibitor; a specific anti-let-7c inhibitor; or both the inhibitors of miR-23a and let-7c. Post-transfection, cells were treated with either no treatment (NT) or PDG (80μg/ml) for 12h, after which IL-6 mRNA levels were measured by qRT-PCR. Treatment of scramble control cells with PDG significantly reduced IL-6 mRNA levels, and these responses were significantly reversed by the presence of both the anti-miR-23a and anti-let-7c inhibitor. *p<0.05 relative to the NT control unless otherwise indicated (n=5).