Human trophoblasts confer resistance to viruses implicated in perinatal infection

Abstract

Objective: Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection?

Study design: Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction.

Results: Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes.

Conclusion: Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

Keywords: chromosome 19 microRNA cluster; conditioned medium; microRNA; trophoblasts; viruses.

Figure 1. Experimental design and model

(A) The medium from primary human trophoblasts (PHT cells) was collected after 48-72 hours, and transferred to nontrophoblast recipient cells 24 h prior to infection. The level of pathogen infection in recipient cells was compared to cells exposed to control medium. See Methods for details. (B) A proposed model for the protective effect of exosome-packaged C19MC miRNAs. Exosomes are transferred from the placenta into the maternal circulation (M), and perhaps into the fetal circulation (F), where they may attenuate viral infection.

Figure 2. PHT conditioned medium or miRNA mimics from the C19MC attenuate infection of select togaviruses

(A) Log scale of Rubella virus titers from cells exposed to non-conditioned or conditioned PHT medium. Data are mean of three independent plaque assays, each run in duplicate. p <0.05 (Student's t test). (B) Activity of alphavirus luciferase reporter constructs. Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Chikungunya virus (CHIKV), and Sindbis virus (SINV), expressing luciferase in Vero cells, were exposed to conditioned PHT or control non-conditioned medium. Data are presented as percent infection relative to control and represent a mean of three independent infections, each run in triplicate. p <0.0001 (ANOVA with Bonferroni correction). (C) EEEV, VEEV, CHIKV, and SINV luciferase expression in cells transfected with miR-517-3p or control scrambled mimic. Data are presented as percent infection relative to control and represent a mean of three independent infections, each run in triplicate. p <0.0001 (ANOVA with Bonferroni correction).

Figure 3. PHT conditioned medium inhibits HIV infection

(A) Relative luciferase activity (RLU) of an HIV Tat-inducible reporter in TZM-bl cells exposed to either conditioned PHT or control non-conditioned medium and infected with increasing concentrations of HIV, as described in Methods. Data are representative of three experiments, each performed in triplicate. * denotes p <0.05, and ** denotes p <0.001 (Student's t test). (B) Relative luciferase expression in TZM-bl cells transfected with miR-517-3p, miR-720, or control scrambled mimic and infected with increasing concentrations of HIV. Data are representative of three experiments, each performed in triplicate. None of the differences were statistically significant.

Figure 4. PHT conditioned medium and miRNA mimics from the C19MC attenuate infection of VZV after initiation of infection and immediate early gene expression

(A) Relative luciferase activity (RLU) from cells infected with either immediate early (IE) reporter virus, or “late” ORF9 reporter virus, exposed to either non-conditioned or conditioned PHT medium. Data are representative of three independent experiments, each performed in triplicate. p <0.001 (Student's t test. (B) Relative luciferase activity (RLU) in MeWo cells transfected with either miR-517-3p, miR-720, or scrambled mimic and infected with VZV_ORF9_Luc. Data are representative of three independent experiments, each performed in triplicate. p <0.05 (ANOVA with Boneferroni correction).

Figure 5. PHT conditioned medium has no effect on Toxoplasma gondii infection

(A) Representative micrographs showing fluorescent parasites within vacuoles in U2OS cells exposed to non-conditioned or conditioned PHT media. Arrow denotes parasitophorous vacuole. Scale bar =10 μM. (B) Vacuole number, or (C) vacuole size in cells exposed to conditioned or non-conditioned media. Quantities are the average of four fields per sample and were assessed via three independent experiments. None of the differences were statistically significant.

Figure 6. PHT conditioned medium has no effect on Listeria monocytogenes replication

Colony forming units for L. monocytogenes after infection of 1.5, 5, and 10 h, as detailed in Methods. Data are presented as percent replication of bacteria in cells exposed to conditioned medium compared to that of bacteria in cells exposed to non-conditioned medium. Data presented are the mean of three independent experiments. None of the differences were statistically significant.