Human placental trophoblasts confer viral resistance to recipient cells

Abstract

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal-fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

Keywords: C19MC; miR-517-3p; primary human trophoblasts.

Fig. 1.

Conditioned PHT medium and exosomes confer viral resistance to recipient cells. (A) PHT or non-PHT cells were infected with a panel of viruses, including coxsackievirus B (CVB), poliovirus (PV), vesicular stomatitis virus (VSV), vaccinia virus (VV), herpes simplex virus-1 (HSV-1), or cytomegalovirus (CMV). Non-PHT cells were as follows: HeLa (CVB, PV), U2OS (VSV, HSV-1, and VV), and human foreskin fibroblasts (HFF; CMV). Shown are the percent infected cells [assessed by immunofluorescence (IF); *P < 0.0001]. (B) Non-PHT recipient cells were exposed for 24 h to nonconditioned or conditioned PHT medium and then infected with CVB, VSV, HCV, or VV. Non-PHT cells were as follows: HFF (CVB), U2OS (VSV, VV), and Huh 7.5 (HCV). Shown are the percent of infected cells [assessed by IF (CVB, VSV), luciferase assay (HCV), or qRT-PCR (VV); *P < 0.05, **P < 0.005]. (C) (Left) Cells were exposed to nonconditioned or conditioned PHT medium for 24 h and then infected with VSV or CVB. (Right) Cells were infected with VSV following exposure to nonconditioned or conditioned PHT medium (*P < 0.05, **P < 0.005). (D) Conditioned PHT medium was subjected to heat inactivation or sonication before 24-h exposure to Vero cells and then infected with VSV. Percent infection assessed as in A (*P < 0.0001). (E) U2OS cells were exposed for 24 h to nonconditioned, conditioned, exosome-depleted conditioned medium, exosomes purified from PHT, JEG-3, or from three preparations of murine dendritic cells (DCs) and then infected with VSV. Percent infection assessed as in A (*P < 0.0005); each PHT exosome preparation was derived from a different placental preparation.

Fig. 2.

PHT and exosomal C19MC miRNAs confer viral resistance to recipient cells. (A) U2OS cells stably expressing control or C19MC BAC were infected with VSV (infection levels assessed by qRT-PCR; *P < 0.0001). (B) U2OS cells were transfected with C19MC miRNA mimics that represent the miRNA subgroups detailed in Table S2 or control mimics and then infected with VSV (shown as percent infected cells assessed by IF; *P < 0.05, **P < 0.001). (C) U2OS cells, transfected with mimics of the six highest expressed C19MC miRNAs, scrambled control, or non-C19MC (miR-1, -424, -630, -720) miRNA mimics, were infected with VSV (infection level assessed by IF or qRT-PCR; *P < 0.0005). (D) U2OS cells, transfected with mimics of the top three antiviral C19MC miRNAs or with scrambled control mimics, were infected with VSV (infection assessed by qRT-PCR; *P < 0.05, **P < 0.0001). (E) U2OS cells, transfected with scrambled control or miR-517b mimic, were infected with VV or HSV-1 (infection assessed as in D; *P < 0.0001).

Fig. 3.

PHT-derived exosomes induce autophagy in recipient cells. (A) U2OS cells transfected with mRFP-LC3b were exposed to nonconditioned, PHT conditioned, or exosome-depleted conditioned PHT medium or purified PHT exosomes for 24 h, and LC3b punctae formation was assessed by confocal microscopy. (Upper) Confocal micrographs. (Lower) Quantification of mRFP-LC3b punctae per cell (*P < 0.0001). (B) (Upper) Electron micrographs of cells exposed to nonconditioned or conditioned PHT medium (Vero), exosome-depleted conditioned PHT medium (Vero), or purified PHT exosomes (U2OS). Arrows denote autophagosomes. (Scale bar, 500 nm.) (Lower) Quantification of electron micrographs of cells exposed to nonconditioned (Vero and U2OS), conditioned PHT media samples (Vero and U2OS), exosome-depleted conditioned medium (Vero), or purified PHT exosomes (U2OS) (*P < 0.0001). (C) U2OS cells transfected with mRFP-LC3b were exposed to nonconditioned or conditioned PHT medium in the absence or presence of 3-MA for 8 h, and LC3b punctae formation was assessed by confocal microscopy. (Upper) Confocal micrographs. (Lower) Quantification of mRFP-LC3b punctae (*P < 0.0005). (D) (Left) Confocal images of VSV entry into U2OS cells transiently transfected with mRFP-LC3b exposed to nonconditioned (Upper) or conditioned (Lower) PHT medium. VSV particles are shown in green and DAPI-stained nuclei are shown in blue (Inset, magnification: 5×). Areas of colocalization appear as yellow. (Right) Quantification of the extent of colocalization between VSV particles and mRFP-LC3B positive punctae (*P < 0.0001).

Fig. 4.

C19MC miRNAs induce autophagy. (A) (Upper) Electron micrographs of U2OS cells transfected with scrambled control or the six highest expressed C19MC miRNA mimics (Table S2). Black arrows denote autophagosomes and/or autolysosomes. (Scale bar, 500 nm.) (Lower) Quantification of electron micrographs shown at top (*P < 0.005) or in PHT cells. (B) U2OS cells were transfected with mRFP-LC3b and either scrambled control or the six highest expressed C19MC miRNA mimics. (Upper) Confocal micrographs. (Lower) Quantification of mRFP-LC3b punctae per cell (*P = 0.0005). (C) (Left) Electron micrographs of U2OS cells transfected with scrambled control or the most potent antiviral miRNA mimics. Black arrows denote autophagosomes and/or autolysosomes. (Scale bar, 500 nm.) (Right) Quantification of adjacent electron micrographs (*P < 0.005). (D) (Upper) A representative immunoblot for p62 or GAPDH in U2OS cells stably transfected with either control Del or C19MC BAC. (Lower) Densitometry of p62 levels (normalized to GAPDH) from three independent immunoblots as described above (*P < 0.05).

Fig. 5.

Suppression of autophagy restores C19MC-medated antiviral effects. (A) U2OS cells transfected with scrambled control or miRNA mimics of the six most prevalent C19MC miRNA mimics. Cells were exposed to 3-MA before and during VSV infection. Relative VSV RNA was analyzed by qRT-PCR (*P < 0.0005). (B) (Left) U2OS cells stably expressing control or C19MC BAC transfected with scrambled control siRNA or beclin-1 siRNA for 72 h were infected with VSV, and relative infection was determined by qRT-PCR. (*P < 0.05, determined using ANOVA with Boneferroni correction). (Right) Immunoblots for beclin-1 or actin in cells transfected as described above. (C) PHT cells were treated with 3-MA before infection with GFP-VSV (in the presence of 3-MA). Relative VSV RNA was analyzed by qRT-PCR (*P < 0.005).