Date of Award

Spring 2022

Rights

Access is available to all users

Document Type

Thesis

Degree Name

Master of Science (MS) in Biology

Department

Biology

Abstract

iv ABSTRACT Effects of GABA on Inflammation and Intestinal Barrier Disruption By Tyrel W. Long Spring 2022 Gut dysbiosis and intestinal barrier disruption have been linked to multiple sclerosis (MS). Our previous works show that experimental autoimmune encephalomyelitis (EAE) induction modifies the gut's microbiota composition, reducing frequencies of gamma-aminobutyric acid (GABA)-producing bacteria. GABA levels are reduced in the brains and circulation of MS patients. We engineered Lactococcus lactis with extra copies of gadB (glutamic acid decarboxylase) and gadC (glutamate/GABA antiporter) to increase GABA levels produced by the bacterium (GAD-L. lactis). EAE studies showed that the treatment with GAD-L. lactis and not with a L. lactis control expressing an empty plasmid (P-L. lactis) reduced the severity of the disease. We hypothesized that the increased levels of GABA produced by GAD-L. lactis would restore the permeability in the intestinal epithelia of EAE mice and in a monolayer composed of Caco-2 cells exposed to inflammatory mediators. Intestinal permeability of the in vivo model was measured by the oral administration of 4-kDa fluorescein isothiocyanate (FITC)-labeled dextran 19 days post-EAE induction. Results showed increased trend of intestinal integrity when EAE with were treated with GAD-L. lactis vs. P-L. lactis (not significant). In vitro, Caco-2 cells were plated on tissue culture trans-well plates creating a monolayer. The Caco-2 cells were exposed to TNF-α, a known barrier disruptor, and to increasing concentrations of GABA (0 – 10 mM). Transepithelial electrical resistance (TER) measurements and the flux of (FITC)-labeled dextran were quantified (0-48 hrs). Our results showed dose- and time-dependent effects of GABA exposure on monolayer integrity. Exposure of cells to 0.5 – 1 mM, but not higher, of GABA resulted in significant increases in monolayer integrity compared with TNF-α controls and unexposed Caco-2 cells over the first (p = 0.0115) and second hour (p = 0.0006). Continuing work with GAD-L. lactis and the P-L. lactis, in conjunction with Caco-2 cell monolayers, will help us understand the role GABA has on permeability.

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