Abstract
Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments.
Highlights
The human vaginal epithelium is coated with a layer of viscoelastic mucus gel comprised of long and heavily glycosylated mucin molecules that are cross-linked, entangled and bundled to form a porous network with large mesh spacings, much larger than antibodies such as IgG and most mammalian viruses
Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens
We have recently shown that IgG that binds to the surface of Herpes Simplex Virus (HSV), including endogenous IgG present in CVM and exogenously added IgG, can trap HSV virions in human CVM at sub-neutralizing concentrations in an Fc- and N-glycan dependent manner [9]
Summary
The human vaginal epithelium is coated with a layer of viscoelastic mucus gel comprised of long and heavily glycosylated mucin molecules that are cross-linked, entangled and bundled to form a porous network with large mesh spacings (average ~340 ± 70 nm [1]), much larger than antibodies such as IgG and most mammalian viruses. We tested the use of Zenon fragments, which bind the Fc region of IgG, gently mixed into CVM to directly label endogenous antibody prior to performing FRAP measurements.
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