Natural Killer Cells Contribute to Pathophysiology of Placenta Leading to Miscarriage in Fetal and Neonatal Alloimmune Thrombocytopenia
Authors: Yougbare Issaka , Wei-she Tai , Zdravic Darko , Chen Pingguo , Zhu Guangheng , Leong-Poi Howard , Dawei Qu , Yu X. Lisa , Lee S. Adamson , Sled John , John Freedman , Ni Heyu 
Published: October 2019

Abstract
Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening disease often leading to severe bleeding diatheses, and/or miscarriage; although the incidence of miscarriage has not been adequately studied. FNAIT is caused by a maternal immune response against fetal platelet antigens, in which >80% reported cases have antibodies against β3 integrin. Maternal antibodies and Natural Killer (NK) cells may target antigen positive trophoblast cells and cause miscarriage, but this hypothesis has never been explored.In this study, we investigated whether platelet antibodies and NK cells impaired trophoblast invasion, and placental angiogenesis and function.
Methods: β3 integrin deficient female mice were immunized with wild-type (WT) platelets and bred with WT males. Placental vascularisation and function were investigated by echography and micro-computered tomography (CT). Angiogenic and inflammatory cytokines, placenta growth factor (PlGF) and fms-like tyrosine receptor levels (Flt-1) were detected by ELISA. Placental function such as nutrient transport was detected by maternal intravenous administration of biotin and its perfusion to the fetus. NK cell phenotype was assessed by FACS. Placenta pathology was investigated by hematoxylin & eosin staining and immuno-histochemistry for cytokeratin-7, NK cell perforin and smooth muscle actin. Apoptosis and decidual remodeling were investigated by TUNEL assays. In vitro, NK cells were co-cultured with trophoblast cell lines and cytotoxicity was detected by flow cytometry.
Results and discussion: Growth restriction and fetal loss/miscarriage only occurred in immunized mothers around embryo day E14.5. Placenta of affected fetuses had significantly reduced vascularization and materno-placental perfusion as demonstrated by ultrasound. CT scan also confirmed shallow development of placental sponge capillaries. These observations are validated by significantly reduced biotin perfusion to the fetuses which had a high hypoxia level. Immunized mice exhibited enlarged spleens as well as an increased Th1 and Th17 immune responses. These pro-inflammatory responses may contribute to trophoblast Flt-1 over-expression and a decreased plasma PlGF/sFlt-1 ratio. E.14.5, the end of organogenesis, is concomitant with trophoblast invasion into spiral arteries. The remodeling of spiral arteries lowers maternal vascular resistance and increases utero-placental blood flow which is critical for healthy pregnancy. Expression of trophoblast marker, Cytokeratin 7, was decreased in the placentas of immunized mice, suggesting scanty invasion invasion. Histology of affected placenta confirmed ischemic tissues, as revealed by significantly reduced numbers of maternal red blood cells in the labyrinth. Unexpectedly, decidual NK cell number remained elevated by day E14.5 in the placenta of immunized mice. Importantly activated NK cells released significant amount of perforin in the placenta, which may destroy target cells expressing β3 integrin. These observations support an abnormal remodeling process mediated by maternal antibodies and NK cells, since increased apoptosis was found in the decidua of immunized pregnant mice compared to naive control. Interestingly, maternal intravenous immunoglobulin therapy ameliorated survival of FNAIT fetuses..
To investigate the molecular and cellular mechanism involved, human choriocarcinoma cells (BeWo) were incubated with both murine anti-β3 antibodies and human HPA-1a IgG. Our data demonstrated, for the first time, that anti-platelet antibodies induced over-expression of the anti-angiogenic molecule, Flt-1 by BeWo cells.
Conclusion: Our data suggest that maternal β3 integrin antibodies and NK cells impaired placental pro-angiogenic signalling and function. Reduced trophoblast invasion may cause poor materno-placental perfusion and fetus loss/miscarriage in FNAIT.

Disclosures
No relevant conflicts of interest to declare.