Date of Graduation

5-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Stenken, Julie A.

Committee Member

Durdik, Jeannine M.

Second Committee Member

Bluhm, Burton H.

Third Committee Member

Lay, Jackson O. Jr.

Fourth Committee Member

Balachandran, Kartik

Keywords

Cytokines; Healthcare-associated infections (HCAIS); Liquid chromatography-mass spectrometry; LL-37; Macrophages; Microdialysis; Quorum sensing

Abstract

The host immune system and bacterial cells are known to interact during the human lifetime. Bacteria secrete a wide variety of signaling molecules, known as quorum sensing (QSC) molecules, that modulate the host immune system. While immune-biofilm interactions involve this chemical signaling network, the mechanisms through which this occurs are not well understood. This work aimed to develop a new method that can be used not only in vitro settings but also in vivo. The microdialysis sampling technique has widely been used in in vitro and in vivo settings in humans, mice, and rats for the collection of neuropeptides, cytokines, and other possible markers. This work focus on the use of microdialysis sampling to study not only the host immune system such as macrophages but also the bacterial biofilm response through QSC during bacterial infection in an in vitro model. Activated macrophages are known to play an important role during bacterial infection because they are phagocytic cells that respond to microenvironment cues using signaling molecules called cytokines.

Several methods were demonstrated to study the cross-communication between macrophage and bacteria-biofilm. First, a method was designed to quantify QSC comprised of ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) with a Bond-elut PPL SPE. Second, an in vitro model, in which macrophage and bacteria/biofilm, were co-culture to measure their respected signaling molecule (QSC for bacteria and/or cytokine for macrophage), and third a microdialysis sampling method was optimized to collect QSC (from bacteria/biofilm) and cytokines (from macrophage). Finally, fourth, a method was developed to study anti-microbial peptide secretion such as LL-37. hCAP-18/LL-37 is a cathelicidin known to be produced by human monocytes (a cationic antimicrobial peptide). This initial work in the detection of LL-37 and the study of the cross-signaling communication between macrophages and bacterial-biofilms provides a fundamental framework for future studies to obtain more information about the host immune system and bacterial interaction in vivo.

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