Human Cytomegalovirus-Induces Cytokine Changes in the Placenta with Implications for Adverse Pregnancy Outcomes

Abstract

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.

Stimulatory effects of human cytomegalovirus tegument protein pp71 lead to increased expression of CCL2 (monocyte chemotactic protein-1) during infection

“…CCL2 mRNA levels were determined using RT-PCR with SYBR Green I One-Step RNA Amplification Master Mix (Roche) on a Roche LightCycler 2.0. PCR primers and cycling conditions were as described previously (Hamilton et al, 2012). Briefly, 25 ng total RNA template (2.5 ml) was added to a 17.5 ml reaction mixture containing 4 ml 56 LightCycler RT-PCR Mix (Roche), 2 mM MgCl 2 (Roche), 0.4 mM each primer (MCP1-3F/MCP1-3R) (Sigma) and 0.4 ml LightCycler RT-PCR Enzyme Mix (Roche).…”

“…Primary infection with CMV is usually asymptomatic in healthy individuals, although infection of immunocompromised patients, including pregnant women, can result in disease (Pass, 2001). Pathogenesis of disease in the infected foetus following maternofoetal transmission likely involves virusinduced upregulation of inflammatory mediators (Hamilton et al, 2012;Scott et al, 2012). CMV encodes proteins with immunomodulatory functions -some of which affect cytokine secretion and chemokine responsiveness, whilst others modulate NK-and T-cell functions, allowing the virus to evade host immune responses (Llano et al, 2003;Michelson, 2004;Penfold et al, 1999;Wilkinson et al, 2008).…”

“…Maternal CMV infection has been demonstrated to upregulate CCL2 and TNF-a in amniotic fluid from mothers of babies born with congenital CMV . The placentae from babies stillborn due to CMV infection also demonstrated intracellular upregulation of pro-inflammatory cytokines, suggesting the increased inflammation may have contributed to the adverse foetal outcome (Hamilton et al, 2012).…”

Therapeutics to prevent congenital cytomegalovirus during pregnancy: what is available now and in the future?

“…These investigations are extremely problematic given potential drug toxicities and the fact that CMV tropism and placental physiology are both highly host-specific. We have therefore begun investigating these experimental compounds in our established ex vivo placental explant model systems 47 …”

Pregnancy, the placenta and Zika virus (ZIKV) infection

“…Studies of infection of explanted placentae in other viral infections such as with CMV show neonatal neural malformation and intra uterine death may be caused partly through Th1 cytokine-induced placental damage 18,19 .…”