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Congenital infection by human cytomegalovirus (HCMV) might result in permanent neurological sequelae, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities. We recently disclosed that Peroxisome Proliferator-Activated Receptor gamma (PPARγ), a transcription factor of the nuclear receptor superfamily, is a key determinant of HCMV pathogenesis in developing brain. Using neural stem cells from human embryonic stem cells, we showed that HCMV infection strongly increases levels and activity of PPARγ in NSCs. Further in vitro experiments showed that PPARγ activity inhibits the neuronogenic differentiation of NSCs into neurons. Consistently, increased PPARγ expression was found in brain section of fetuses infected by HCMV, but not in uninfected controls. In this commentary, we summarize and discuss our findings and the new insights they provide on the neuropathogenesis of HCMV congenital infection.
The pathogeny of infectious diseases dysregulates a variety of cellular pathways, which identification may shed new light about their role in the healthy cell. In particular, infections by neurotropic viruses in pregnancy may lead to neurodevelopmental abnormalities in the fetus and point out novel mechanisms contributing to neurogenesis. Particularly relevant from this point of view is congenital infection by Human Cytomegalovirus (HCMV), as it is the most frequent infectious cause of permanent neurological sequelae.
HCMV is a BetaHerpes virus with a worldwide distribution. It is highly prevalent in the general population: seroprevalence ranges between 40 and 90%, with the greatest values among racial/ethnic minorities and persons of lower socio-economic background status.1 HCMV is transmitted through close non-sexual or sexual contact, breastfeeding, blood transfusions, and organ transplantation.2 For the pregnant woman, the most likely source of infection is the contact with the urine or saliva of young children, including her own children.2 No vaccine is available so far. Lifelong latency is established after a primary infection. Though infection of immunocompetent adults is almost always benign, HCMV is responsible for serious illness and death in immunocompromised hosts, and is a major hazard for the fetus after infection during pregnancy. HCMV infection in utero is believed to occur through transplacental hematogenous spread.3 It affects 0.5–2 % of live births, whereas approximately 10 % of infected newborns are symptomatic at birth, and up to 15 % of those asymptomatic at birth develop delayed HCMV-related disease manifestations in their early years.4 Congenital HCMV disease is associated with a wide range of neurodevelopmental disabilities, including hearing and vision loss and mental retardation, as well as structural brain abnormalities including intracranial calcifications, microcephaly, hydrocephalus, ventriculomegaly, ventriculomegaly, polymicrogyria, porencephaly, and schizencephaly.5 Neurological outcomes are more severe when infection occurs during the first trimester.5
Deciphering HCMV tropism is the brain was critical to investigate its neuropathy. Founder studies in the mouse revealed that HCMV murine counterpart, namely murine cytomegalovirus (MCMV), infected the developing brain the cerebral ventricular walls, a region known to contain neural progenitors.6 Strikingly, infected cells seemed to migrate from the (sub-) ventricular zones to the cortical plate or the hippocampus.6 Mouse neurons were also found to be sensitive to infection. Studies in human were limited for obvious reasons, and in vitro studies were performed with primary cultures of human brain cells prepared from deceased, uninfected fetus. Brain microvascular endothelial cells, astrocytes, neuronal cells, oligodendroglial cells, microglia/macrophages, and neural progenitor/stem cells were found to be sensitive to HCMV.5 However, no histological data identifying the different cell types actually infected in utero during congenital HCMV infection were available, except histopathological analysis of postmortem brain samples which revealed HCMV inclusion bodies in the brain.7 In our study, we explored the expression of the immediate early HCMV antigen (IE), a factor encoded by the HCMV genome and critical for virus replication, in histopathological slides from deceased infected or non-infected fetus.8 In agreement with the studies in the mouse model, we observed cells clearly immunoreactive to IE in the ependymal and germinative zones of the brain of infected cases. However, no labeling was found in the white matter, whereas, as expected, brain vessels were positive to IE. These findings disclosed that neural progenitors and ependymal cells were the preferential, if not the only, neural cells targeted by HCMV during fetal brain infection.
Based on the assumption that HCMV infection is likely to disturb neural progenitor homeostasis or differentiation, a number of studies investigated the ability of neural progenitors infected in vitro by HCMV to generate neurons or astrocytes. These studies used progenitors which were prepared from brain from deceased, uninfected fetus, and eventually infected in vitro and driven to differentiate. They showed sometimes conflicting results, and, at least, revealed considerable diversity in the phenotype of such progenitors following HCMV infection. Indeed, HCMV infection of neural progenitors was found to (1) inhibit self-renewal and proliferation, inhibit neuronal differentiation, and induce apoptosis,9 or (2) inhibit astrocyte differentiation,10 or (3) cause premature and abnormal differentiation into an uncharacterized cell type,11 or (4) reduce the number of proliferating CD24-expressing progenitors.12 Two other studies used neural stem cells generated from human induced pluripotent stem (iPS) cells and reported that HCMV infection impaired neuronal differentiation.13,14
In order to perform molecular investigations on the outcomes of HCMV infection on neural progenitor cells, we used a new model, highly neuronogenic neural stem cells from embryonic stem cells (NSCs) (Fig. 1). NSCs were generated through early neuroepithelial differentiation of human ES cells in a monolayer system using 2 SMAD inhibitors (SB431542, Noggin) and the defined medium N2B27.15 This method allowed for efficient neural commitment and avoided possible instrumental factors as donor variability (including gestation age), use of batch-dependent components, and feeder cell conditioned medium. NSCs showed self-renewal and continuous growth in defined conditions without the need of generating neurospheres. They displayed a cortical phenotype with positive immunostaining and/or high levels of expression of polarized neural stem cells and radial glia markers such as nestin, GFAP, BLBP, SOX2, PAX6, POU3F3, NRCAM and PARD3, in addition to genes involved in corticogenesis (MEF2C, BDNF, RTN4, ODZ1, FLNA and PAFAH1B1), in the absence of immunoreactivity to non-cortical markers.16 This phenotype was particularly relevant with respect to the fact that congenital HCMV infection targets cortical areas of the developing brain as well as radial glia cells.17 NSCs showed ability to differentiate into neurons positive for the markers HUC/D and β-III tubulin upon growth factor removal. On this basis, we investigated the outcomes of infection on neuronogenic differentiation of NSCs. Consistently with the works cited above, we found that NSCs were permissive to HCMV infection (Fig. 2) and that infection dramatically impaired NSC differentiation into neurons.8 In the search for the molecular bases of such inhibition of differentiation, we next investigated the levels and activity of the nuclear receptor PPAR gamma (PPARγ) in infected NSCs.
PPARγ is a host-encoded transcription factor with pleiotropic roles in metabolism, inflammation, cell differentiation and migration. Notably, brain development abnormalities were reported in PPARγ−/− and PPARγ−/+mice embryos.18 Various, and sometimes discordant, effects of PPARγ agonists were also observed in a variety of neural cell models (reviewed in ref. 19). Together those findings suggested that PPARγ could play a role in brain development. Incidentally, we had previously showed that PPARγ activity is mandatory for HCMV replication and is dramatically enhanced in cytotrophoblasts and placentae infected by HCMV.20 Therefore, we investigated the outcomes of HCMV infection on PPARγ in NSCs.
Uninfected NSCs showed weak expression of PPARγ as assessed by immunofluorescence analysis, western blot, and luciferase reporter assays.8 Conversely, HCMV infection strongly increased PPARγ levels and activity in NSCs, as shown by immunofluorescence analysis, western blot, luciferase reporter assays and chromatin immunoprecipitation.8 Notably, we found that infected NSCs exerted a bystander effect on neighboring uninfected cells, which also showed enhanced PPARγ levels. Levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARγ agonist, were significantly increased in infected NSC cultures. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARγ activity. However, these results did not demonstrate that PPARγ activation in infected NSCs was a causative factor for impaired differentiation. Hence we designed and performed a number of experiments to determine whether PPARγ activation was necessary and/or sufficient to impair neuronogenesis in vitro. Thus we demonstrated that (1) pharmacological activation of PPARγ ectopically expressed through lentiviral transduction under the control of the EF1α minimal promoter 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.8 These findings revealed that PPARγ activation was necessary and sufficient to impair NSC differentiation into neurons. Last, we explored the relevance of these findings in vivo, through immunohistological examination of brain slices from a set of infected (N = 20) or uninfected control (N = 5) fetus of different gestational ages. Strinkingly, nuclear PPARγ was immunodetected in the brain germinative zones of congenitally infected fetuses, but in none of the control samples8 (Fig. 3), what strongly supported the role of PPARγ in the disease phenotype.
Our findings revealed that PPARγ is a key determinant of the pathogeny of HCMV congenital infection. Further, they demonstrated that NSCs are a relevant tool which fairly recapitulates disease mechanisms in vitro. Hence, this cell platform should also help investigating the molecular and cellular pathogenic bases of other neurotropic viruses, such Zika virus (ZIKV), especially given that NSCs are sensitive to ZIKV infection in vitro.21 Last, our findings reveal a key role for PPARγ in neurogenesis and in the pathophysiology of HCMV congenital infection. Identification of PPARγ gene targets in the infected brain is critical to decipher the genetic and molecular events leading to impaired neurogenic differentiation in NSCs with abnormal activation of PPARγ (should they be infected or not). In the long term, dissecting the PPARγ signaling pathways will provide new insight on neuronogenesis in health and disease.
No potential conflicts of interest were disclosed.