Characterization of 3D organotypic epithelial tissues reveals tonsil-specific differences in tonic interferon signaling

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Abstract

Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e. , cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data ( https://viz.datascience.arizona.edu/3DEpiEx/ ).

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  1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The current study uses 3D organotypic rafts to culture primary keratinocytes from Foreskin, Tonsil and Cervix. Further the authors looked at the transcriptomic profiles of each tissue types to study similarities and differences depending on the tissue of origin as well as show the similarity in the tissue specific gene signatures and the ex-vivo samples (data from GTEx). As mentioned by authors Skin and Cervix keratinocytes have been previously cultured on collagen rafts however extending it to Tonsil provides resource and possibility of growing more tissue specific epithelial cells in 3D.

    Majo…

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    Referee #3

    Evidence, reproducibility and clarity

    This is very carefully analysed and written study describing the transcriptional differences between in vitro models of epithelia derived from cervix, foreskin and tonsil tissues. Importantly, they compare the findings to in vivo samples using publicly available data. The findings are significant and will be of interest to the scientific community. I cannot fault the analysis pathways or the conclusions, and the manuscript is a pleasure to read. I recommend it is accepted for publication as is.

    Significance

    This is an important study that is highly significant for researchers interested in epithelia tissue and infection. The data …

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Jackson et al.'s manuscript describes an experiment that directly compares 3D organotypic assays created with primary human epithelial cells from foreskin, cervix and tonsil using histological and bulk RNA sequencing approaches. The authors convincingly show the retention of site-specific histological and transcriptomic differences between the stratified epithelial tissues in culture. Differentially expressed genes are identified and pathway analyses suggest genes that might be involved in the different differentiation processes between these tissue sites and differential regulation of ECM and immune pathways. Differentially …

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    The current study uses 3D organotypic rafts to culture primary keratinocytes from Foreskin, Tonsil and Cervix. Further the authors looked at the transcriptomic profiles of each tissue types to study similarities and differences depending on the tissue of origin as well as show the similarity in the tissue specific gene signatures and the ex-vivo samples (data from GTEx). As mentioned by authors Skin and Cervix keratinocytes have been previously cultured on collagen rafts however extending it to Tonsil provides resource and possibility of growing more tissue specific epithelial cells in 3D.

    Major comments

    1. As the papers focus is to …