Exosomes from Ureaplasma parvum-infected ectocervical epithelial cells promote feto-maternal interface inflammation but are insufficient to cause preterm delivery

Abstract

This study determined if exosomes from ectocervical epithelial (ECTO) cells infected with Ureaplasma parvum (U. parvum) can carry bacterial antigens and cause inflammation at the feto-maternal interface using two organ-on-chip devices, one representing the vagina-cervix-decidua and another one mimicking the feto-maternal interface, and whether such inflammation can lead to preterm birth (PTB). Exosomes from U. parvum-infected ECTO cells were characterized using cryo-electron microscopy, nanoparticle tracking analysis, Western blot, and Exoview analysis. The antigenicity of the exosomes from U. parvum-infected ECTO cells was also tested using THP-1 cells and our newly developed vagina-cervix-decidua organ-on-a-chip (VCD-OOC) having six microchannel-interconnected cell culture chambers containing cells from the vagina, ectocervical, endocervical, transformation zone epithelia, cervical stroma, and decidua. The VCD-OOC was linked to the maternal side of our previously developed feto-maternal interface organ-on-a-chip (FMi-OOC). Cell culture media were collected after 48 h to determine the cytokine levels from each cell line via ELISA. For physiological validation of our in vitro data, high-dose exosomes from U. parvum-infected ECTO cells were delivered to the vagina of pregnant CD-1 mice on E15. Mice were monitored for preterm birth (PTB, < E18.5 days). Exosomes from ECTO cells infected with U. parvum (UP ECTO) showed significant downregulation of exosome markers CD9, CD63, and CD81, but contained multiple banded antigen (MBA), a U. parvum virulence factor. Monoculture experiments showed that exosomes from UP ECTO cells delivered MBA from the host cell to uninfected endocervical epithelial cells (ENDO). Moreover, exposure of THP-1 cells to exosomes from UP ECTO cells resulted in increased IL-8 and TNFα and reduced IL-10. The OOC experiments showed that low and high doses of exosomes from UP ECTO cells produced a cell type-specific inflammatory response in the VCD-OOC and FMi-OOC. Specifically, exosomes from UP ECTO cells increased pro-inflammatory cytokines such as GM-CSF, IL-6, and IL-8 in cervical, decidual, chorion trophoblast, and amnion mesenchymal cells. The results from our OOC models were validated in our in vivo mice model. The inflammatory response was insufficient to promote PTB. These results showed the potential use of the VCD-OOC and FMi-OOC in simulating the pathophysiological processes in vivo.

Effects of Ureaplasma parvum infection in the exosome biogenesis‐related proteins in ectocervical epithelial cells

“…Cryo‐EM images showed circular membrane‐bound vesicles (Figure S1). Based on our previous study, U. parvum infection did not significantly change the size and concentrations of exosomes from ECTO cells 26 . We employed NanoLC–MS/MS analysis to perform a proteomic analysis.…”

“…A previous study in Rhesus macaques showed the U. parvum only induced mild uterine inflammation without overt chorioamnionitis 24 . Several animal studies also showed that ascending infection with U. parvum was not associated with high preterm birth rates 25,26 . These mechanisms often lead to persistent mild infections with mycoplasmas 27 …”

“…The mechanism by which U. parvum is transmitted from mother to fetus, that is, from the lower reproductive tract to the amniotic cavity, amniotic fluid, and fetal tissues, remains unclear as the intact cervix is a major barrier, and Ureaplasma infection alone is not highly immunogenic or causes cell death to facilitate its spread 7,23 . In previous studies, the propagation via extracellular vesicles as a means of infecting the developing fetus has been explored and postulated that Ureaplasma may control extracellular vesicle (specifically exosomes of 30−160 nm) biogenesis to package themselves for delivery in other cells 21,26 . Based on these observations, we hypothesized that U .…”

“…However, in a mouse model, this inflammation was insufficient to cause preterm birth. 26 Another study also showed that mycoplasma proteins could be packaged in exosomes released by infected tumor cells and modulate immune cell functions in the host. 33 These findings suggest the involvement of exosome biogenesis in the intracellular infection, survival, and propagation of infection in mycoplasmas, including U. parvum.…”

“…24 Several animal studies also showed that ascending infection with U. parvum was not associated with high preterm birth rates. 25,26 These mechanisms often lead to persistent mild infections with mycoplasmas. 27 The cervix is one of the tissues susceptible to persistent infections with genital mycoplasmas.…”