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.

Figure 1. MCP-1 and TNF-α expression is elevated in CMV-infected placentae from stillborn babies.

(A) MCP-1 and TNF-α expression in uninfected (CMV−/Other−), other microorganism-infected (CMV−/Other+) and CMV-infected (CMV+/Other−) placentae from stillborn babies. Data presented as box plots with median value, Q1, Q3 and range. Significant differences between groups were determined with the Mann-Whitney U test and threshold for significance adjusted to account for multiple comparisons using Bonferroni’s correction; *p<0.0167, **p<0.003. (B) Representative images of immunohistochemical localisation of MCP-1 and TNF-α protein (red staining) in uninfected and CMV-infected placental tissue from stillborn babies. In chorionic villi, cytokine localised in syncytiotrophoblast (st) and cytotrophoblast (ct) cells, mesenchymal stromal cells (msc) and endothelial cells of fetal capillaries (ec). Scale bars represent 70 µm.

Figure 2. CMV productively infects placental villous explants.

Productive AD169 and Merlin infection of villous explants was determined by staining for CMV immediate early/early (IE/E), early/late (pp65) and late (gB) protein. Representative images are of AD169 and Merlin infected villous explants 12 days post inoculation. Scale bars represent 50 µm.

Figure 3. CMV actively replicates in cytotrophoblast and mesenchymal cells, but not syncytiotrophoblasts, of placental villous explants.

CMV laboratory strain AD169 and wild-type Merlin infected syncytiotrophoblasts (CK7+/VIM−/hPL+), cytotrophoblasts (CK7+/VIM−/hPL−) and mesenchymal cells of the villous stroma (CK7−/VIM+/hPL−) as determined by staining for CMV immediate early/early protein. Active replication (CMV IE/E+, pp65+) was observed in both cytotrophoblasts and mesenchymal cells but not syncytiotrophoblasts (CMV IE/E+, pp65−) (inserts and arrow heads). Representative images are of 4 µm consecutive histological sections of AD169 infected villous explants 12 days post inoculation. No difference in cellular tropism was observed between AD169 and Merlin strains. Scale bars represent 100 µm.

Figure 4. CMV infection is significantly greater in AD169- compared with Merlin-infected placental villous explants.

(A) Representative images of CMV immediate early/early (IE/E) antigen detection in AD169- (left) and Merlin-infected (right) placental villous explants 12 days post inoculation (CK7; Cytokeratin-7). Scale bar represents 70 µm. (B) Number of cells per mm² of villous explant tissue expressing CMV IE/E protein in AD169- compared with Merlin-infected explants. (C) No significant differences in villous explant area (mm²) were observed between the CMV-infected explant groups (p = 0.1). Data presented as box plots with median value, Q1, Q3 and range. Significant differences between groups (denoted as *) were determined using a one-tailed Spearman’s correlation.

Figure 5. CMV infection of placental villous explants results in upregulation of MCP-1 and TNF-α expression.

(A) MCP-1 and TNF-α expression in mock-infected, AD169-infected (0.2 pfu/ml) and Merlin-infected (0.02 pfu/ml) villous explants. Data presented as box plots with median value, Q1, Q3 and range. Significant differences between groups were determined with the Mann-Whitney U test and threshold for significance adjusted to account for multiple comparisons using Bonferroni’s correction; *p<0.025, **p<0.005 and ***p<0.0005. (B) Correlation between degree of CMV placental explant infection with corresponding MCP-1 and TNF-α expression. MCP-1 and TNF-α protein expression was plotted against the number of cells expressing CMV immediate early/early (IE/E) protein per mm² of AD169 and Merlin infected villous explant tissue. Significant correlations (denoted by *) were determined by two-tailed nonparametric spearmans correlation with trend plotted as straight lines.