Infection and upregulation of proinflammatory cytokines in human brain vascular pericytes by human cytomegalovirus

Abstract

BackgroundCongenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood–brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported.MethodsPrimary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated ‘SBCMV’. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry.ResultsHCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6 compared to both mock-infected as well as heat-killed virus controls. A 6.6-fold induction of IL-6 and no induction TNF-alpha was observed in SBCMV-infected cell supernatants at 24 hours postinfection. Using archival brain tissue from a patient coinfected with HCMV and HIV, we also found evidence of HCMV infection of pericytes using dual-label immunohistochemistry, as monitored by NG2 proteoglycan staining.ConclusionHCMV lytic infection of primary human brain pericytes suggests that pericytes contribute to both virus dissemination in the CNS as well as neuroinflammation.

Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

“…During JEV infection, the permeability-inducing effect of IL-6 was supported by the finding of the inhibitory effect of IL-6 neutralizing antibody against JEV-conditioned medium-induced barrier disruption and the barrier-disrupting effect of exogenous recombinant IL-6. The corresponding compromised endothelial barrier integrity by infected pericytes and accompanying IL-6 production was also noted in cases of HIV-1 and human cytomegalovirus infection (22,23). In addition to macrophage-derived neutrophil chemotactic factor (15), our findings suggest that IL-6 released by brain microvascular endothelium neighboring pericytes is of paramount importance in Japanese encephalitis-associated BBB breakdown.…”

“…The regulation of BBB integrity by pericytes is of increasing interest due to the fact that these cells are in close proximity to brain endothelium and release a large number of endothelial permeability-regulating molecules. Pericytes are known to secrete elevated levels of permeability-inducing factors such as TNF-␣, IL-1␤, IL-6, metalloproteinases, and VEGF under different conditions (19)(20)(21)(22)24). These factors were elevated in JEV-infected animals and cultured glial cells (10,11,15,31,(37)(38)(39).…”

“…Brain pericytes, the nearest neighbors of brain microvascular endothelial cells sharing a common basal membrane in cerebral capillaries, have a regulatory effect on BBB integrity (19)(20)(21). Virus-infected or stressed pericytes produced elevated levels of proinflammatory cytokines and compromised the integrity of the BBB in vitro (22)(23)(24). Although the viruses can be detected in BBB endothelial cells after systemic infection (17), the results of a brain microvascular endothelial cell monoculture model study showed that the increased vascular permeability during JEV infection could not solely be produced by endothelial infection (25).…”

Human cytomegalovirus infection of human adipose-derived stromal/stem cells restricts differentiation along the adipogenic lineage

“…This raises interesting questions in regards to a possible stromal progenitor cell reservoir for latent HCMV or at the very least an amplification reservoir. 46 Sundin et al 33 show that ex vivo-expanded hMSCs from HCMV seropositive individuals displayed no signs of viral infection as measured by PCR. ASCs are regarded as immunoprivileged and their residence in the body's largest endocrine organ would be an ideal site for HCMV to influence host health.…”

The Association Between Biliary Atresia and Cytomegalovirus Hepatitis

“…This protein represents the largest component in virus structure 14 . The expression of this protein coincides with viral ly c replica on 15 , also the expression of this protein refl ect the ac ve viral infec on 16 .…”