Cytomegalovirus Infection Triggers the Secretion of the PPARγ Agonists 15-Hydroxyeicosatetraenoic Acid (15-HETE) and 13-Hydroxyoctadecadienoic Acid (13-HODE) in Human Cytotrophoblasts and Placental Cultures

<div><p>Introduction</p><p>Congenital infection by human cytomegalovirus (HCMV) is a leading cause of congenital abnormalities of the central nervous system. Placenta infection by HCMV allows for viral spread to fetus and may result in intrauterine growth restriction, preeclampsia-like symptoms, or miscarriages. We previously reported that HCMV activates peroxisome proliferator-activated receptor gamma (PPARγ) for its own replication in cytotrophoblasts. Here, we investigated the molecular bases of PPARγ activation in infected cytotrophoblasts.</p><p>Results</p><p>We show that onboarded cPLA₂ carried by HCMV particles is required for effective PPARγ activation in infected HIPEC cytotrophoblasts, and for the resulting inhibition of cell migration. Natural PPARγ agonists are generated by PLA₂ driven oxidization of linoleic and arachidonic acids. Therefore, using HPLC coupled with mass spectrometry, we disclosed that cellular and secreted levels of 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE) were significantly increased in and from HIPEC cytotrophoblasts at soon as 6 hours post infection. 13-HODE treatment of uninfected HIPEC recapitulated the effect of infection (PPARγ activation, migration impairment). We found that infection of histocultures of normal, first-term, human placental explants resulted in significantly increased levels of secreted 15-HETE and 13-HODE.</p><p>Conclusion</p><p>Our findings reveal that 15-HETE and 13-HODE could be new pathogenic effectors of HCMV congenital infection They provide a new insight about the pathogenesis of congenital infection by HCMV.</p></div

Name and abbreviation of each metabolite quantified in our study.

<p>Name and abbreviation of each metabolite quantified in our study.</p

Retention time (RT), line equation, coefficient of determination (r2), maximum percent residual, limit of detection (LOD), limit of quantification (LOQ) and attributed internal standard (IS) for each metabolite.

<p>Retention time (RT), line equation, coefficient of determination (r2), maximum percent residual, limit of detection (LOD), limit of quantification (LOQ) and attributed internal standard (IS) for each metabolite.</p

Onboarded cPLA₂ is required for PPARγ activation and inhibition of cytotrophoblast migration.

<p>(A) TLC analysis of bodipy-phosphatidylcholine (B-PC) incubated in the presence of buffer (mock), purified PLA₂ (PLA2), native HCMV particles (HCMV), HCMV particles pre-treated by MAFP (HCMV+MAFP) or UV-irradiated (HCMV-UV). (B) PPARγ activity luciferase assay performed with HIPEC in various conditions, using a reporter plasmid responsive to PPARγ (pGL4-PPRE) or a control plasmid (PGL4), 48 h pi or post treatment onset. HCMV: HIPEC infected by live HCMV particles; vi: the viral inoculum was treated beforehand by MAFP; cm: control with 50 nM MAFP in the culture medium; GW: PPARγ inhibitor GW9662; Rosi: PPARγ agonist rosiglitazone. RLU: relative luciferase units; **: p < 0.01 (Kruskal-Wallis test). The assay was repeated twice.(C) Wound-healing assays. HCMV+MAFP: the viral inoculum was treated beforehand by MAFP; HCMV+GW: the HIPEC were infected in the presence of GW9662; HCMV-UV: the viral inoculum was UV-irradiated. Results are expressed as the percent variation relative to the control. The figure shows results of a representative experiment, out of two independent experiments, each comprising triplicate measures. NI: non-infected.</p

Increased amounts of 15-HETE and 13-HODE secreted by from early placenta explants infected by HCMV.

<p>(A) LC-MS/MS analysis of the amounts of PUFA-derived lipids secreted from histocultures from 12 first trimester placentas either infected by HCMV (CMV) or uninfected (NI). *: p < 0.05; **: p < 0.01 (Wilcoxon test). (B) Immunostaining analysis of HCMV antigen IE in infected (HCMV) or uninfected (NI) placental explants cultured ex vivo. Representative views are shown. Note the positive, nuclear IE staining in the cytotrophoblast layer (ST) surrounding the villus stroma (S).</p

Optimal conditions for lipid separation by LC-MS/MS.

<p>* Deuterated compound used as an internal standard.</p><p>Optimal conditions for lipid separation by LC-MS/MS.</p

Proposed model of PPARγ activation in HCMV infection of placental cells.

<p>HCMV particles (HCMV) carry onboarded cPLA2 (oPLA2) which catalyses linoleic acid (LA) and arachidonic acid (AA) release from host membrane phospholipids (PL). AA and LA undergo oxidization driven by 15-lipoxygenase (15-LOX), which generates 15HETE and 9-HODE, respectively. 15HETE and 9-HODE are activating ligands of PPARγ, which dimerizes with RXR to regulate the expression of the host and virus genomes, resulting in impaired migration abilities in vitro, and enhanced IE transcription and viral replication. M: cell membrane, C: cytoplasm, N: nucleus.</p

PPARγ Is Activated during Congenital Cytomegalovirus Infection and Inhibits Neuronogenesis from Human Neural Stem Cells

<div><p>Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1% of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses and investigated the outcomes of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARγ), a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARγ levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARγ agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARγ activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARγ was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV-infected NSCs with the PPARγ inhibitor T0070907 restored a normal rate of differentiation. The role of PPARγ in the disease phenotype was strongly supported by the immunodetection of nuclear PPARγ in brain germinative zones of congenitally infected fetuses (N = 20), but not in control samples. Altogether, our findings reveal a key role for PPARγ in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARγ gene targets in the infected brain.</p></div

Nuclear PPARγ expression in germinative zone of HMV-infected human fetal brains.

<p>Shown are representative results of immunohistological staining of brain sections from fetuses infected by HCMV (A-G) or from controls (H, I) using antibodies against PPARγ (A-E; F, left; G-I) or IE (F, right). The reference number of each donor is indicated at the bottom left of each panel. Clinical details are summarized in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005547#ppat.1005547.t001" target="_blank">Table 1</a>. PPARγ positive cells (arrows) are detected in the germinative, periventricular, areas and in ependyma (double arrow) in cases, but not in controls. Insets show the localization of the optical field within the brain sections (arrowheads). Note the nuclear localization of PPARγ (A-G), the presence of PPARγ positive cell islets surrounding one IE positive cell in two fields from serial sections (F) and clusters of PPARγ immunoreactive cells around lesional tissue (G). Scale bar: 50 μm.</p

NSCs are permissive to HCMV infection.

<p>(A) Immunofluorescence analysis of NSCs infected by live (HCMV) or UV-irradiated (HCMV+UV) HCMV, or uninfected (NI), showing nuclear staining to the HCMV Immediate Early antigen (IE) two days post infection (dpi) at a multiplicity of infection (MOI) of 10. DAPI staining and merged pictures are shown. Scale bar: 50 μm. (B) Top: automated counting of immunofluorescence data showing increasing numbers of IE-positive NSCs over time in cultures infected by live HCMV at a MOI of 1 or 10, but not in cultures infected by UV-irradiated HCMV or in uninfected cultures. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. Bottom: western blot analysis showing increasing levels along time of the 72 and 86 kDa isoforms of IE in infected NSCs (MOI 10). (C) Western blot analysis showing production of the early and late HCMV antigens UL44 and pp28, respectively, in infected NSCs (MOI 10), at 8 days pi. (D) Top: transmission electron microscopy of NSC cultures infected by HCMV (MOI 10), showing a cytomegalic NSC (arrowhead) and lipid vesicles (arrows), close to two morphologically normal NSCs (NI), and HCMV particles adsorbed onto the cell surface (inset). Scale bar: 5μm or 0.2 μm (inset). Bottom: transmission electron microscopy of the cytoplasm of an infected NSC, revealing mature viral particles (arrowheads) and dense bodies (asterisks). Pictures were taken 6 days after infection. Scale bar: 0.5μm. (E) Titration of viral particles present in the supernatants of infected NSCs (MOI 10). Supernatants were harvested at different times pi (horizontal axis) and were titrated on MRC5 fibroblasts. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. Virus strain was AD169 except for panel A (VHL/E).</p