Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Prodger, Jessica L.

Abstract

The foreskin is a site of HIV-1 acquisition in heterosexual males. The lack of relevant in vitro models that mimic the foreskin microenvironment, including innate immune and barrier functions, has limited our understanding of susceptibility at this site. We hypothesize that we can establish organotypic in vitro foreskin that mimics in vivo tissue. Organotypic foreskin models were generated and are composed of stratified cell layers that express E-cadherin suprabasally, filaggrin apically, and expressed TLR1, TLR2, and β-defensin-1 mRNA similar to in vivo foreskin. Preliminary results suggest organotypic foreskin tissues retain the ability to respond to LPS. Lastly, organotypic foreskin permeability appears high when subject to mechanical agitation. This work has established culture techniques to generate multi-layer epithelia and the methods necessary to characterize innate immune and barrier functions. This sets the foundation for future work to fully develop the model, including integrating immune cells, the microbiota, or HIV-1.

Summary for Lay Audience

The foreskin creates a fold of skin that provides an environment where bacteria intolerant to oxygen (known as anaerobes) can live. These anaerobes have been linked to the risk of acquiring human immunodeficiency virus (HIV) in heterosexual men. Our group has shown that anaerobes may increase HIV risk by causing local foreskin inflammation (an immune response) while disrupting foreskin permeability. This could allow HIV to penetrate the foreskin and infect immune cells (HIVs target cell type) that have gathered in the area due to this inflammation.

To discern how bacteria mechanistically alter susceptibility to HIV, we propose creation of an artificial foreskin that mimics natural foreskin in structure and function. Cells will be isolated from donor foreskins and grown on top of one another to generate layers of the skin. The artificial foreskin will be characterized next to the donor foreskin to see if it successfully mimics important aspects of the skin, such as the ability to detect and respond to bacteria on its surface and form a barrier to the outside environment.

In this study, artificial foreskins were generated that mimicked the structure of the natural foreskin, such as being composed of multiple cell layers stacked on top of one another. Tissues also expressed structural proteins commonly found in the natural foreskin such as E-cadherin (helps attach cells together) and filaggrin (helps in developing and maintaining the outer barrier of the skin). Similar to natural foreskin, artificial foreskins maintained gene expression of microbe sensors Toll-like Receptor 1 and 2, and antimicrobial protein β-defensin-1. Preliminary data suggests they may be able to respond to stimulation with a bacterial ligand. Lastly, data suggests that mechanical shaking during tissue development results in disorganized organotypic foreskin tissue structure with increased permeability.

This body of work has established culture conditions to isolate and propagate primary keratinocytes from adult foreskin tissue and use these to generate organotypic foreskin tissues. These tissues show multilayer stratification and differentiation, and expression of innate immune molecules and barrier proteins. These important developmental steps lay the groundwork for full model characterization, eventual incorporation of anaerobes, and challenge with HIV-1.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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