A role for uric acid and the Nalp3 inflammasome in antiphospholipid antibody-induced IL-1β production by human first trimester trophoblast

Abstract

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta2-glycoprotein I (β2GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β2GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β2GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β2GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.

Figure 1. Antiphospholipid antibodies induce trophoblast IL-1β processing and secretion.

Trophoblast cells were either not treated (NT) or treated with (i) the anti-β₂GPI mAb, IIC5 (20 µg/ml) or mouse IgG1 control (mIgG; 20 µg/ml); or (ii) human aPL-IgG (500 µg/ml) or normal human IgG control (hIgG; 500 µg/ml) for 72 hrs. (A) Barcharts show levels of secreted IL-1β as determined by ELISA. Data are from 5 independent experiments. *p<0.01 versus the NT control. (B) Trophoblast cells were evaluated for active IL-1β (17 kDa) expression by Western blot (representative blots are shown). Barcharts below show quantification of protein expression as determined by densitometry and normalized to β-actin. Data are from 3 independent experiments. *p<0.05 versus the NT control.

Figure 2. Antiphospholipid antibody-induced trophoblast IL-1β is dependent upon caspase-1.

Trophoblast cells were either not treated (NT) or treated with the anti-β₂GPI mAb, IIC5 (20 µg/ml), all in the presence of media or the caspase-1 inhibitor (5 µM) for 72 hrs. (A) Active IL-1β (17 kDa) expression was evaluated by Western blot. (B) Barchart shows IIC5-induced IL-1β secretion as determined by ELISA and expressed as fold change relative to the untreated control. Data are from 4 independent experiments (*p<0.05).

Figure 3. Antiphospholipid antibody-induced trophoblast IL-1β is dependent on ASC and Nalp3.

Trophoblast transfected to express either (A & C) shRNA for ASC (sh-ASC) or a control sequence (sh-control) (n = 5); or (B & D) shRNA for Nalp3 (sh-Nalp3) or a Nalp3 mutated targeting sequence (Nalp3-mut) (n = 4), were either not treated (NT) or treated with the anti-β₂GPI mAb, IIC5 (20 µg/ml) for 72 hrs. Culture supernatants were measured for (A & B) IL-1β and (C & D) IL-8 by ELISA. *p<0.05; **p<0.001 versus the NT control, unless indicated otherwise.

Figure 4. Antiphospholipid antibodies induce trophoblast uric acid production by trophoblasts via TLR4, which in turn triggers IL-1βproduction.

(A) Trophoblast cells were either not treated (NT) or treated with (i & ii) the anti-β₂GPI mAb, IIC5 (20 µg/ml) or mouse IgG1 control (mIgG; 20 µg/ml) for 24, 48 and 72 hrs; or (iii) aPL-IgG (500 µg/ml) for 72 hrs. Cell lysates (intracellular) and supernatants (secreted) were measured for uric acid. Data are from 4–6 independent experiments. *p<0.05; **p<0.001 versus the NT control. (B) Trophoblast cells were pretreated with either media or the TLR4 antagonist, LPS-RS (10 µg/ml) for 30 mins before cells were then treated with or without the anti-β₂GPI mAb, IIC5 (20 µg/ml) for 72 hrs. Barcharts show levels of IIC5-induced: (i) secreted uric acid and (ii) secreted IL-1β, expressed as fold change relative to the untreated control. Data are from 4 independent experiments (*p<0.01). (C) Trophoblast cells were treated with or without the anti-β₂GPI mAb, IIC5 (20 µg/ml) in the presence of media or uricase for 72 hrs. Barchart shows levels of IIC5-induced IL-1β secretion, expressed as fold change relative to the untreated control. Data are from 3 independent experiments (*p<0.05).