Epithelial cells along the mucosal surface provide the front line of host defense against pathogen infection in the gastrointestinal (Gl) tract. Because of the importance of Toll-like receptor (TLR) signaling in the initiation and regulation of Gl mucosal immunity, the overall objective of this application is to better understand the molecular mechanisms by which TLR signaling coordinates Gl epithelial antimicrobial defense. Our preliminary studies demonstrate that microbial challenge stimulates exosome release from the apical side of cultured Gl epithelial monolayers in a TLR4-dependent manner. Released exosomes shuttle a variety of antimicrobial peptides and display antimicrobial activity ex vivo. Moreover, activation of TLR4/NF-?B signaling causes alterations in expression of microRNAs (miRNAs), small non-coding miRNAs that regulate gene expression at the posttranscriptional level. Selected TLR4-responsive miRNAs may target effector molecules that regulate the exocytotic process and, thus, are potentially involved in TLR4-mediated exosome release. Based on these exciting novel preliminary data, we propose to test the hypothesis that the release of exosomes from epithelial cells is regulated by TLR signaling with the involvement of miRNA- mediated gene regulation at the posttranscriptional level, and that it contributes to TLR-mediated Gl epithelial antimicrobial defense. We will use in vitro and in vivo infection models and complementary biochemical, molecular, and morphologic approaches to test four interrelated Specific Aims: i) The TLR signaling pathway regulates release of apical exosomes from epithelial cells in response to microbial challenge;ii) TLR signaling stimulates exosome release from epithelial cells through the IKK/SNAP-23- associated exocytotic process with the involvement of miRNA-mediated posttranscriptional regulation;iii) TLR signaling regulates exosomal shuttling of antimicrobial peptides;and iv) epithelial exosomes contribute to TLR-mediated epithelial antimicrobial defense. The proposal is conceptually innovative as it tests new concepts regarding TLR-mediated mucosal antimicrobial defense. The information obtained from this study should provide a rational basis for the design and implementation of new therapeutic strategies.

Public Health Relevance

The proposed research will study the role of epithelial cell-derived apical exosomes and miRNA-mediated posttranscriptional gene regulation in TLR-associated epithelial antimicrobial immune responses. The overall goal is to elucidate the molecular mechanisms by which TLR signaling coordinates epithelial antimicrobial defense and provide a basis for the identification of novel new targets for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI095532-04
Application #
8683088
Study Section
Special Emphasis Panel (ZAI1-WFD-I (M2))
Program Officer
Rothermel, Annette L
Project Start
2011-07-01
Project End
2016-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
4
Fiscal Year
2014
Total Cost
$287,012
Indirect Cost
$88,388
Name
Creighton University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
053309332
City
Omaha
State
NE
Country
United States
Zip Code
68178
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