Placental Inflammation and Fetal Injury in a Rare Zika Case Associated With Guillain-Barré Syndrome and Abortion

Abstract

Zika virus (ZIKV) is an emerging virus involved in recent outbreaks in Brazil. The association between the virus and Guillain-Barré syndrome (GBS) or congenital disorders has raised a worldwide concern. In this work, we investigated a rare Zika case, which was associated with GBS and spontaneous retained abortion. Using specific anti-ZIKV staining, the virus was identified in placenta (mainly in Hofbauer cells) and in several fetal tissues, such as brain, lungs, kidneys, skin and liver. Histological analyses of the placenta and fetal organs revealed different types of tissue abnormalities, which included inflammation, hemorrhage, edema and necrosis in placenta, as well as tissue disorganization in the fetus. Increased cellularity (Hofbauer cells and TCD8⁺ lymphocytes), expression of local pro-inflammatory cytokines such as IFN-γ and TNF-α, and other markers, such as RANTES/CCL5 and VEGFR2, supported placental inflammation and dysfunction. The commitment of the maternal-fetal link in association with fetal damage gave rise to a discussion regarding the influence of the maternal immunity toward the fetal development. Findings presented in this work may help understanding the ZIKV immunopathogenesis under the rare contexts of spontaneous abortions in association with GBS.

Keywords: Guillain-Barré syndrome; Zika virus; fetal infection; histopathology; immune response.

Figure 1

Histopathological analysis of the placenta and detection of ZIKV. (A–C) Placenta of a non-ZIKV patient stained with H.E. and presenting normal features: membrane (Mem), (MB) basal membrane, (FL) fibroblastic layer, (CL) compact layer, (Ep) epithelium, chorionic villi (CV), maternal decidua (Dec), and blood vessels (BV). (D–K) Sections of ZIKV-infected placental tissue stained with H.E., showing abnormalities in membrane, with cellular degeneration (CD), in the decidua and chorionic villi, including fibrinoid necrosis (FN), hemorrhage (He), mononuclear inflammatory infiltrate (Inf), infarct (IFT), calcification (Ca) and Hofbauer cells with clear cytoplasm (Hf). (L,M,P,Q) The flavivirus E protein and NS1 antigens of ZIKV were not detected by immunohistochemistry in the control placenta. (N–O) Detection of ZIKV E protein in decidual cells (DC), cytotrophoblasts (CTB), mesenchymal cells (MS) and Hofbauer cells (Hf) of the infected placenta. (R–T) The NS1 protein of ZIKV was also detected by immunohistochemistry in decidual cells (DC), cytotrophoblasts (CTB) and Hofbauer cells (Hf).

Figure 2

Characterization of mononuclear cell subpopulations in ZIKV-infected placental tissue, colocalization with virus and cytokine-producing cell profile. (A,B) Detection of CD68⁺ cells (Hofbauer cells) by immunohistochemistry in decidua and chorionic villi of control placenta, respectively. (C,D) Hofbauer cells in decidua and chorionic villi of ZIKV infected placental tissue, respectively. (F,G) Detection of CD8⁺ cells by immunohistochemistry in decidua and chorionic villi of control placenta, respectively. (H,I) CD8⁺ cells immunostained in decidua and chorionic villi of ZIKV-infected placental tissue, respectively. (E,J) Quantification of CD68⁺ and CD8⁺ cells in ZIKV case and control, respectively. (K,L) Colocalization by immunofluorescence of the NS1 protein (fluorescent green) and CD11b for identification of leukocytes (fluorescent red). Nuclei were stained using DAPI (fluorescent blue). (K) ZIKV NS1 antigen was not detected in the control placenta. (L) Cells presenting dual staining (green and red) were observed in the ZIKV-infected placenta. (M,N). Detection of TNF-α in cells of chorionic villi of control and ZIKV infected placenta by immunohistochemistry, respectively. (P,Q) Production of IFN-γ in cells of membrane of control and ZIKV infected placenta, respectively. (S,T) VEGFR2-expressing cells of decidua in control and ZIKV infected placenta, respectively. (V,X) Detection of CCL5/RANTES in cells of chorionic villi of control and ZIKV infected placenta, respectively. (O,R,U,Z) Quantification of the number of cells expressing TNF-α, IFN-γ, VEGFR2, and CCL5/RANTES, in ZIKV case and control, respectively. Asterisks indicate differences that are statistically significant between groups (^***p < 0.001).

Figure 3

Histopathological analysis of the fetal organs. (A–J) All tissues were stained with H. E. (A) Brain of a non-ZIKV case presenting normal aspect: cerebral cortex (CC) and neurons (Ne). (B) Brain of a ZIKV infected fetus, presenting areas of degenerated nerve fibers (DF). (C) Lung section of a control fetus showing normal bronchioles (BC). (D) Injuries in fetal lung infected by ZIKV: disorganized bronchioles (DBC) with loss of cylindrical appearance, focal areas of hyaline membrane (HM), diffuse mononuclear infiltrates (Inf) and peeled cells of respiratory epithelium (PC). (E) Skin dermis (DM) of a non-ZIKV case presenting normal aspect, epidermis (Epi), blood vessel (BV). (F) Skin dermis of a ZIKV infected fetus, with perivascular and mononuclear infiltrate (PInf and Inf) and areas of edema (E). (G) Kidney of a non-ZIKV case presenting normal aspect, with normal glomerulus (GR) and proximal contorted tubules (PCT). (H) Kidney sections showing injuries, including: disorganized renal glomerulus (GR), tubular disarrangement and inflammatory infiltrate (Inf). (I) Section of a control liver, with normal bile duct (BD) and portal vein (PV). (J) Liver of a ZIKV infected fetus, presenting dilatation of sinusoidal capillaries (SC), hepatic parenchymal disorganization and Kupffer cells (KC).

Figure 4

Detection of ZIKV antigens in fetal organs. (B,D,F,H,J,L,N,P,R,T) The flaviviral E and ZIKV-NS1 protein were detected in all tissues studied. (B,L) Microglial cells and neurons of the brain tissue were positive for ZIKV E protein and NS1, respectively. (D,N) ZIKV E and NS1 proteins were detected in alveolar macrophages. (F,P) Section of a skin dermis showed mononuclear cells of an inflammatory infiltrate and endothelial cells stained respectively to ZIKV E and NS1 proteins. (H,R) ZIKV E protein and NS1 detection in macrophages of the kidney. (J,T) Detection of ZIKV E protein and NS1 in hepatocytes, respectively. (A,C,E,G,I,K,M,O,Q,S) The E and NS1 antigens of ZIKV were not detected by immunohistochemistry in the control fetus.