Aspectos imunohistopatológicos e ultraestruturais de placentas de pacientes infectadas por ZIKV na gestação e investigação dos efeitos desta infecção em mastócitos e células trofoblásticas in vitro

Abstract

Zika fever is an arbovirus, transmitted mainly by mosquitoes of the genus Aedes, but there is also the possibility of vertical transmission, with alarming reports of Congenital Zika Syndrome cases associated with infection. It is known that the Zika virus (ZIKV) crosses the placenta and infects the fetus, but the pathogenesis of this disease has not yet been clarified. From this, the present study aimed to better understand the consequences that ZIKV infection causes on human placental tissue and tissues of a stillbirth, as well as on the HMC-1 mast cell and HTR-8/SVneo trophoblastic cell lines. The tissues were subjected to histopathological analysis, immunoassays and in situ hybridization for viral, immune cells and inflammatory cytokines/mediators detection, in addition to the ultrastructural assessment of placental cells. RT-PCR real-time and PRNT assays were performed with the patient's serum. In cell lines, An infection protocol was standardized after in vitro kinetics assay using immunofluorescence and / or flow cytometry. Then, we analyzed the ultrastructural changes of infected cells by transmission electron microscopy, observed the degranulation and release of cytokines in mast cells or oxidative stress in placental cells. Histological damage was found in the placentae, such as villous immaturity, fibrin deposition, inflammatory infiltrate, areas of calcification and decreased extracellular matrix components. We detected positive cells for the ZIKV antigens, proving even the replication with the NS1 protein and negative RNA labeling in different placental cells and resident or circulating immune cells. The increase in cellularity (CD68+ cells and T CD8+ lymphocytes) and the expression of local proinflammatory cytokines such as IFN-ɣ and TNF-α, and other mediators, such as RANTES/CCL5, MMPs and VEGFR-2, support the character of inflammation and dysfunction placental disease caused by the virus. In addition, we noted that the decrease in BDNF neurotrophin can modulate fetal neuronal damage, since its expression was lower in the placentae of patients who had babies with microcephaly. Histopathological changes, virus detection and evidence of replication were also observed in all stillborn organs analyzed. The ultrastructural aspects of infected cells in both tissues and cell cultures showed changes in the organelles, mainly in the endoplasmic reticulum and mitochondria. We observed that mast cells are permissive to infection in placental tissues and in vitro, and the infection stimulates the degranulation and release of cytokines and mediators. The infected trophoblastic cells showed a significant increase in the activity of the antioxidant enzyme SOD, in the reactive species MDA and NO, while the enzymatic activity of CAT was lower in cells infected by ZIKV. With our findings, we can affirm that the placental changes caused by ZIKV are not pathognomonic, however, this infection leads to severe placental changes, and the infection can results in fetal impairment or damage. The findings presented in this work contribute to understand the immunopathogenesis of Zika in the maternal-fetal context.