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Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the inner leaflet of the plasma membrane (IPM) is low in unstimulated cells and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial. Here we report a systematic and rigorous evaluation of basal IPM cholesterol levels in a wide range of mammalian cells with different properties employing cholesterol sensors derived from the D4 domain of the Perfringolysin O toxin and a sterol-transfer protein, Osh4. Results consistently showed that, although basal IPM cholesterol levels vary significantly among cells, they remain significantly lower than cholesterol levels in the outer leaflets. We found that IPM cholesterol levels were particularly low in all tested primary cells. These results support the universality of the low basal IPM cholesterol concentration under physiological conditions. We also report here the presence of sequestered IPM cholesterol pools, which may become available to cytosolic proteins under certain physiological conditions. We hypothesize that these pools may partly account for the low basal level of available IPM cholesterol. In conclusion, we provide new experimental data that confirm the asymmetric transbilayer distribution of the plasma membrane cholesterol, which may contribute to regulation of various cellular signaling processes at the plasma membrane.

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however, whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production, we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) coculture model. RSV infection of BEC/HLF cocultures led to decreased hyaluronidase expression by HLFs, increased accumulation of HA, and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however, BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared with control mice, which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared with SPC-Cre(-) RSV-infected littermates. Taken together, these data demonstrate that altered extracellular matrix accumulation of HA occurs following RSV infection and may contribute to airway inflammation. In addition, loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment.