Inhibition of The Auto-inflammation Suppressor Protein ISG15 Triggers Preeclampsia by Blocking Trophoblast Migration and Invasion
Objective: Covalent binding of proteins (aka ISGylation) by interferon-induced ubiquitin-like modifier 15 protein (ISG-15) negatively regulates IFN-α/β responses. In humans, homozygous loss-of-function mutations in the ISG15 gene suppresses interferon (IFN)-γ production in lymphocytes and induces an abnormally hyper-IFN-α/β mediated immune response causing severe auto-inflammation. Elevated plasma and decidual interleukin (IL)-6 expression and impaired decidual IFN signaling are associated with preeclampsia (PE). Using microarray analysis, we found that IL-6 inhibits ISG15 expression in primary cytotrophoblast (CTB) cultures. Our in situ analysis showed that interstitial CTBs in PE vs. control placentas exhibit significantly lower ISG15 immunoreactivity. This study investigated the role of ISG15 on CTB migration and invasion.
Materials and Methods: Immunohistochemistry was performed on term decidua basalis specimens to determine cell specific ISG15 expression (n=4). Migration and invasion assays were performed on normal and ISG15-siRNA silenced HTR8/SVneo cells (human first-trimester chorionic villi explant-derived immortalized cytotrophoblasts) using non-coated and matrigel coated trans-wells and 24-h wound healing assays in 12-well culture plates. Statistical analysis used student’s t-test.
Results: Among decidua basalis cells, immunostaining revealed the highest in situ ISG15 expression in interstitial CTBs. Immunoblotting and qRT-PCR of HTR8 cultures confirmed significantly reduced ISG15 protein and mRNA levels following ISG15 siRNA vs. control siRNA treatment. Phase-contrast microscopy observed flattened and larger cells in HTR8 cultures following ISG15 siRNA vs. control siRNA treatment. Moreover, ISG15 siRNA silencing reduced HRT8 migration (Mean ± SEM 0.410±0.05 vs. 0.679± 0.080; p = 0.026; n=6), invasion (0.373±0.02 vs. 0.573±0.06; p= 0.024; n=5) and delayed in vitro wound healing compared to control siRNA treatment at 4 to12 h.
Conclusion: These results indicate that ISG15 expression levels are crucial for trophoblast morphology and function (migration/invasion). By blocking trophoblast invasion, reduced ISG15 levels could contribute to impaired spiral artery transformation that reduces utero-placental blood flow in PE. Thus, induction of ISG15 expression is likely to be therapeutic in preeclampsia.
Supported by U01HD087213 by NICHD.
Materials and Methods: Immunohistochemistry was performed on term decidua basalis specimens to determine cell specific ISG15 expression (n=4). Migration and invasion assays were performed on normal and ISG15-siRNA silenced HTR8/SVneo cells (human first-trimester chorionic villi explant-derived immortalized cytotrophoblasts) using non-coated and matrigel coated trans-wells and 24-h wound healing assays in 12-well culture plates. Statistical analysis used student’s t-test.
Results: Among decidua basalis cells, immunostaining revealed the highest in situ ISG15 expression in interstitial CTBs. Immunoblotting and qRT-PCR of HTR8 cultures confirmed significantly reduced ISG15 protein and mRNA levels following ISG15 siRNA vs. control siRNA treatment. Phase-contrast microscopy observed flattened and larger cells in HTR8 cultures following ISG15 siRNA vs. control siRNA treatment. Moreover, ISG15 siRNA silencing reduced HRT8 migration (Mean ± SEM 0.410±0.05 vs. 0.679± 0.080; p = 0.026; n=6), invasion (0.373±0.02 vs. 0.573±0.06; p= 0.024; n=5) and delayed in vitro wound healing compared to control siRNA treatment at 4 to12 h.
Conclusion: These results indicate that ISG15 expression levels are crucial for trophoblast morphology and function (migration/invasion). By blocking trophoblast invasion, reduced ISG15 levels could contribute to impaired spiral artery transformation that reduces utero-placental blood flow in PE. Thus, induction of ISG15 expression is likely to be therapeutic in preeclampsia.
Supported by U01HD087213 by NICHD.
