Dissertation

Elucidating the Mechanisms of Immune Regulation by Bacillus subtilis

Zamora-Pineda, Jesus

2022

Overview

Probiotics are a class of microorganisms can offer a health benefit to the host. How these microorganisms alter the host remains a mystery. The scientific group of Dr. Katherine L. Knight, Ph.D. studies the probiotic bacterium Bacillus subtilis. Previous reports from the Knight Laboratory showed that B. subtilis can prevent disease caused by the colonic pathogen Citrobacter rodentium. This protection is mediated by the production of exopolysaccharide (EPS). Intraperitoneal administration of purified EPS is sufficient to protect mice from colitis disease. Furthermore, EPS is protective in disease models of sepsis, allergic eosinophilia, and graft versus host disease, indicating that EPS from B. subtilis has anti-inflammatory properties. In this dissertation, we determined the chemical and structural characteristics of EPS and how EPS can generate an anti-inflammatory response. Our results showed that EPS is a complex carbohydrate with a composition of arabinose, mannose, glucose, galactose, N-acetyl glucosamine, and N-acetyl galactosamine. The backbone sugar is decorated with arabinosyl oligosaccharides that by themselves have lymphocyte inhibitory activity. EPS can generate inhibitory bone marrow-derived dendritic cells that inhibit pan- T cell activation. This inhibition is mediated by the activation and cross-linking of the Toll-like receptor 4 (TLR4), which initiates the activation of the canonical NF-kB pathway. The transcription factors in this pathway transcribe the immune-suppressive enzyme indolamine 2,3-dioxygenase (IDO), and by using ido1-/- mice and IDO inhibitors, we showed that IDO was required for EPS inhibition of T cell proliferation. IDO metabolizes the essential amino acid tryptophan into kynurenine metabolites, and depletion of tryptophan in the microenvironment and the formation of kynurenines lead to the inhibition of T cell activation. Furthermore, IDO expression induces the formation of the IDO-Kynurenine-AhR loop which further extends the anti-inflammatory response of EPS. By examining how EPS induces an anti-inflammatory response through TLR4, we found that EPS does not internalize CD14, a TLR4 co-receptor of LPS, but instead EPS internalizes the receptors CD11b and Dectin-1. Both CD11b and Dectin-1can shape TLR4 signaling into an anti-inflammatory response while blocking the pro-inflammatory branch. We hypothesize that EPS acts through these receptors in conjunction with TLR4. Data in this thesis demonstrate that carbohydrate from a gram-positive bacterium activates TLR4 on dendritic cells and induces expression of IDO, which inhibits T cell proliferation. By identifying and understanding active molecules from probiotics, a new class of therapeutics to treat and prevent diseases will likely become available.

• Publisher: ProQuest Dissertations & Theses
• Subject: Immunology / Microbiology
• ISBN: 9798352962916