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

Mulla MJ, Salmon JE, Chamley LW, Brosens JJ, Boeras CM, Kavathas PB, Abrahams VM - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Maternal-Fetal Medicine, Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, United States of America.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675211&req=5

pone-0065237-g001: 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.

Mentions: IL-1β secretion can only occur after pro-IL-1β is processed into its active form. This is commonly mediated by the Nalp3 inflammasome; a complex of Nalp3 and ASC that subsequently activates caspase-1, leading to pro-IL-1β processing [24]. We, therefore, examined whether our previous observation of aPL-mediated IL-1β secretion by the trophoblast [16] was associated with its processing. As shown in Figure 1A, the first trimester trophoblast cell line, Sw.71, secreted significantly elevated levels of IL-1β: (i) in response to the mouse anti-β2GPI mAb, IIC5; and (ii) in response to patient-derived aPL-IgG. The mouse and human IgG controls had no effect on trophoblast IL-1β secretion (Figure 1A; i & ii). After treatment with the mouse anti-β2GPI mAb, IIC5, but not the mouse IgG control (mIgG), expression of the cleaved active form of IL-1β (17 kDa) significantly increased, as determined by Western Blot analysis (Figure 1B; i). We also observed a decrease in the endogenous expression of the pro-form of IL-1β (31 kDa) after treatment with IIC5, when compared to the NT and mouse IgG controls. However, because of a high level of background this was difficult to quantify (data not shown). Similarly, treatment of trophoblast cells with human aPL-IgG, but not the human IgG control, significantly increased expression of active IL-1β (Figure 1B; ii). Because of high background caused by the aPL-IgG and IgG control preparations, we were unable to clearly see the pro-IL-1β band (data not shown). Having established trophoblast IL-1β processing and secretion in response to aPL, we sought to determine the role of caspase-1. The presence of a caspase-1 inhibitor significantly reduced IIC5-induced IL-1β processing (Figure 2A), and IIC5-induced IL-1β secretion (Figure 2B).


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

Mulla MJ, Salmon JE, Chamley LW, Brosens JJ, Boeras CM, Kavathas PB, Abrahams VM - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675211&req=5

pone-0065237-g001: 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.
Mentions: IL-1β secretion can only occur after pro-IL-1β is processed into its active form. This is commonly mediated by the Nalp3 inflammasome; a complex of Nalp3 and ASC that subsequently activates caspase-1, leading to pro-IL-1β processing [24]. We, therefore, examined whether our previous observation of aPL-mediated IL-1β secretion by the trophoblast [16] was associated with its processing. As shown in Figure 1A, the first trimester trophoblast cell line, Sw.71, secreted significantly elevated levels of IL-1β: (i) in response to the mouse anti-β2GPI mAb, IIC5; and (ii) in response to patient-derived aPL-IgG. The mouse and human IgG controls had no effect on trophoblast IL-1β secretion (Figure 1A; i & ii). After treatment with the mouse anti-β2GPI mAb, IIC5, but not the mouse IgG control (mIgG), expression of the cleaved active form of IL-1β (17 kDa) significantly increased, as determined by Western Blot analysis (Figure 1B; i). We also observed a decrease in the endogenous expression of the pro-form of IL-1β (31 kDa) after treatment with IIC5, when compared to the NT and mouse IgG controls. However, because of a high level of background this was difficult to quantify (data not shown). Similarly, treatment of trophoblast cells with human aPL-IgG, but not the human IgG control, significantly increased expression of active IL-1β (Figure 1B; ii). Because of high background caused by the aPL-IgG and IgG control preparations, we were unable to clearly see the pro-IL-1β band (data not shown). Having established trophoblast IL-1β processing and secretion in response to aPL, we sought to determine the role of caspase-1. The presence of a caspase-1 inhibitor significantly reduced IIC5-induced IL-1β processing (Figure 2A), and IIC5-induced IL-1β secretion (Figure 2B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Maternal-Fetal Medicine, Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, United States of America.

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.

Show MeSH
Related in: MedlinePlus