Date of Award

Fall 9-4-2025

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

First Advisor

KATHERINE KNIGHT

Abstract

Exopolysaccharides (EPS) secreted by the commensal bacterium Bacillus subtilis exhibit potent anti-inflammatory properties, yet the underlying molecular mechanisms remain incompletely understood. We hypothesized that EPS reprograms antigen-presenting cells toward a tolerogenic state by modulating TLR4–MyD88 signaling. To test this, we began using a chemically defined, high-yield EPS production and purification protocol that largely eliminates protein and DNA contaminants, thus improving functional reproducibility across experiments using this new method. In vitro, EPS engaged TLR4 on murine dendritic cells (DC2.4) without inducing classical NF-κB activation, seen by LPS stimulation. Rather, EPS treatment upregulated inhibitory molecules, PD-L1 and PD-L2 while leaving activation markers, CD80/CD86 expression unchanged. These data are consistent with a regulatory phenotype in this cell type. Functionally, EPS-conditioned DC2.4 cells suppressed antigen-independent proliferation of both CD4⁺ and CD8⁺ T lymphocytes. EPS pre-treatment also induced a state of endotoxin tolerance, where subsequent LPS challenge failed to induce IL-6 secretion or NF-κB p65 phosphorylation. In vitro, a single dose of EPS conferred robust protection against LPS-induced systemic inflammation, maintaining suppressed IL-6 levels for at least three days Interestingly, this tolerogenic effect was not observed in primary bone marrow derived dendritic cells and macrophages, thus, suggesting a context or cell-type specific mechanism for such induced tolerance. These findings identify B. subtilis EPS as a safe and novel TLR4- dependent immunoregulatory molecule. We believe EPS induces tolerance by decoupling microbial sensing receptors from inflammatory signaling and output. This work provides both mechanistic insight and a scalable production method to support the development of microbial polysaccharides as next-generation anti-inflammatory therapeutics.

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