Antiphospholipid Antibodies Inhibit Trophoblast Toll-Like Receptor and Inflammasome Negative Regulators.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. Although these antibodies are heterogeneous, some anti-β2 -glycoprotein I (anti-β2 GPI) antibodies can activate Toll-like receptor 4 (TLR-4) and NLRP3 in human first-trimester trophoblasts. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL-induced trophoblast inflammation. Human trophoblasts were not treated or were treated with anti-β2 GPI aPL or control IgG in the presence or absence of the common TAM (TYRO3, AXL, and Mer tyrosine kinase [MERTK]) receptor ligand growth arrest-specific protein 6 (GAS6) or the autophagy-inducer rapamycin. The expression and function of the TAM receptor pathway and autophagy were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Antiphospholipid antibody-induced trophoblast inflammation was measured by qRT-PCR, activity assays, and ELISA. Anti-β2 GPI aPL inhibited trophoblast TAM receptor function by reducing cellular expression of the receptor tyrosine kinases AXL and MERTK and the ligand GAS6. The addition of GAS6 blocked the effects of aPL on the TLR-4-mediated interleukin-8 (IL-8) response. However, the NLRP3 inflammasome-mediated IL-1β response was not affected by GAS6, suggesting that another regulatory pathway was involved. Indeed, anti-β2 GPI aPL inhibited basal trophoblast autophagy, and reversing this with rapamycin inhibited aPL-induced inflammasome function and IL-1β secretion. Basal TAM receptor function and autophagy may serve to inhibit trophoblast TLR and inflammasome function, respectively. Impairment of TAM receptor signaling and autophagy by anti-β2 GPI aPL may allow subsequent TLR and inflammasome activity, leading to a robust inflammatory response.

Mulla MJ ，Weel IC ，Potter JA ，Gysler SM ，Salmon JE ，Peraçoli MTS ，Rothlin CV ，Chamley LW ，Abrahams VM ... -  《-》

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》

Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA. aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05). While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL. These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs. This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests. N/A.

Gysler SM ，Mulla MJ ，Guerra M ，Brosens JJ ，Salmon JE ，Chamley LW ，Abrahams VM ... -  《-》

Antiphospholipid antibodies induce a pro-inflammatory response in first trimester trophoblast via the TLR4/MyD88 pathway.

Mulla MJ ，Brosens JJ ，Chamley LW ，Giles I ，Pericleous C ，Rahman A ，Joyce SK ，Panda B ，Paidas MJ ，Abrahams VM ... -  《-》

Modulation of trophoblast function by concurrent hyperglycemia and antiphospholipid antibodies is in part TLR4-dependent.

While diabetes and APS are individually associated with increased risk of poor perinatal outcomes, in particular preeclampsia, recent studies have demonstrated an association between concurrent aPL and diabetes leading to an increased risk of pregnancy morbidity. Hyperglycemia and aPL have independently been shown to alter human trophoblast function by inducing a pro-inflammatory, anti-angiogenic, and antimigratory response. However, little is known about the effects of concurrent hyperglycemia and aPL on trophoblast function. A human first-trimester extravillous trophoblast cell line was exposed to glucose at 5 mmol/L (normoglycemia) or 25 mmol/L (hyperglycemia), all in the presence or absence of low-dose aPL or control IgG. For some experiments, the TLR4 antagonist, LPS-RS, was included. Cell culture supernatants were measured for inflammatory IL-1β and IL-8, and angiogenic PlGF, sFlt-1, and sEndoglin by ELISA. Inflammasome-associated uric acid was measured using a bioassay; caspase-1 was measured using an activity assay. Trophoblast migration was quantified using a two-chamber colorimetric assay. Compared to excess glucose alone, combination excess glucose and low-dose aPL (a) further augmented trophoblast inflammatory IL-1β, inflammasome-associated uric acid and caspase-1, and pro-angiogenic PlGF; (b) dampened trophoblast inflammatory IL-8, anti-angiogenic sEndoglin, and sFlt-1; and (c) further reduced trophoblast migration. Our findings indicate that while concurrent aPL and hyperglycemia are overall detrimental to trophoblast function, the presence of two simultaneous insults triggers some protective effects.

Leon-Martinez D ，Mulla MJ ，Han CS ，Chamley LW ，Abrahams VM ... -  《-》