Single- and double-stranded viral RNA generate distinct cytokine and antiviral responses in human fetal membranes

Abstract

There has been growing interest in the role of viral infections and their association with adverse pregnancy outcomes. However, little is known about the impact viral infections have on the fetal membranes (FM). Toll-like receptors (TLR) are thought to play a role in infection-associated inflammation at the maternal-fetal interface. Therefore, the objective of this study was to characterize the cytokine profile and antiviral response in human FMs exposed to viral dsRNA, which activates TLR3, and viral ssRNA, which activates TLR8; and to determine the mechanisms involved. The viral dsRNA analog, Poly(I:C), induced up-regulated secretion of MIP-1α, MIP-1β, RANTES and TNF-α, and down-regulated interleukin (IL)-2 and VEGF secretion. In contrast, viral ssRNA induced a broader panel of cytokines in the FMs by up-regulating the secretion of IL-1β, IL-2, IL-6, G-CSF, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α and GRO-α. Using inhibitory peptides against TLR adapter proteins, FM secretion of MIP-1β and RANTES in response to Poly(I:C) was MyD88 dependent; MIP-1α secretion was dependent on MyD88 and TRIF; and TNF-α production was independent of MyD88 and TRIF. Viral ssRNA-induced FM secretion of IL-1β, IL-2, IL-6, G-CSF, MIP-1α, RANTES and GRO-α was dependent on MyD88 and TRIF; MIP-1β was dependent upon TRIF, but not MyD88; and TNF-α and MCP-1 secretion was dependent on neither. Poly(I:C), but not ssRNA, induced an FM antiviral response by up-regulating the expression of IFNβ, myxovirus-resistance A, 2',5'-oligoadenylate synthetase and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G. These findings demonstrate that human FMs respond to two viral signatures by generating distinct inflammatory cytokine/chemokine profiles and antiviral responses through different mechanisms.

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators

“…Furthermore, we have previously shown using siRNA studies that engagement of TLR2 and TLR5 enhances production of cytokines and MMP-9 in fetal membranes [22]. In vitro studies have also shown that bacterial products such as LPS and flagellin, and the viral dsRNA analogue poly(I:C), stimulate pro-inflammatory cytokines in fetal membranes [22,27,58]. Thus, we used LPS, flagellin and poly(I:C) to mimic infection-induced preterm birth in vitro to assess the importance of AMPK in regulating pro-inflammatory cytokines in fetal membranes.…”

KLF5 regulates infection- and inflammation-induced pro-labour mediators in human myometrium

“…Associations between viral infections and chorioamnionitis and/or spontaneous preterm birth have been also reported (Gomez et al 2008, Tsekoura et al 2010. Furthermore, the viral dsRNA analogue poly(I:C) induces pro-inflammatory cytokines in foetal membranes (Bakaysa et al 2014). It was thus of interest to determine if KLF5 regulates pro-inflammatory and pro-labour mediators in the presence of IL1b, flagellin and the poly(I:C) in human myometrium.…”

“…NFkB has been shown to regulate the expression of numerous pro-labour genes including IL6, IL8 and COX2 in the presence of infection or inflammation (Belt et al 1999, Lappas et al 2002, 2003, Lappas & Rice 2007, Bakaysa et al 2014. Studies in non-gestational tissues demonstrate that NFkB is required for KLF5 signalling.…”

Viral Single Stranded RNA Induces a Trophoblast Pro-Inflammatory and Antiviral Response in a TLR8-Dependent and -Independent Manner1

“…Total RNA from the trophoblast cell lines and mouse placental tissue was extracted using TRIzol, chloroform, and 2-propanol according to the manufacturer's instructions (Life Technologies) as previously described [35,36]. Total RNA concentrations were measured at A260 using a NanoDrop (Thermo Scientific) and stored at À808C.…”

“…Total RNA concentrations were measured at A260 using a NanoDrop (Thermo Scientific) and stored at À808C. For the analysis of mRNA expression levels, quantitative real-time PCR (qPCR) was performed using the KAPA SYBR FAST qPCR kit (Kapa Biosystems) on the Bio-Rad CFX Connect Real-Time System (Bio-Rad) as previously described [35][36][37]. All primer sequences used are shown in Table 1.…”