Small molecules modulators of Toll-like receptor 4 (TLR4): Biological activity and mechanistic insight into fibrosis and immune signaling

Fibrosis is a major global health problem responsible for up to 45% of deaths in industrialized countries. It results from maladaptive wound healing characterized by persistent myofibroblast activation, excessive extracellular matrix (ECM) accumulation, and organ dysfunction, as seen in diseases such as idiopathic pulmonary fibrosis (IPF) and intestinal fibrosis in Crohn’s disease (CD). Despite advances in understanding fibrosis pathophysiology, effective treatments remain limited. Toll-like receptor 4 (TLR4), a crucial receptor of innate immunity expressed in fibroblasts and stromal cells, acts as a central mediator of fibrotic responses by linking immune activation with fibroblast-driven ECM deposition. Activation of TLR4 by damage-associated molecular patterns (DAMPs) such as fibronectin EDA and tenascin-C promotes fibroblast activation and a profibrotic feedback loop. Pharmacological inhibition and genetic deletion of TLR4 have shown promise in reducing fibrosis, suggesting TLR4 as an attractive therapeutic target. In this thesis, we analyzed the expression changes of TLR4 and its co-receptor MD-2 using published scRNAseq datasets and the TAMMA database in fibrotic versus healthy control tissues, focusing on both lung and intestinal fibrosis. Additionally, we investigated the impact of transforming growth factor beta 1 (TGF-β1) on lung fibroblasts isolated from wild-type (WT) and Tlr4 knockout (Tlr4-/-) mice to assess the role of this receptor in idiopathic pulmonary fibrosis (IPF) progression and validate our rationale. Then, two novel small-molecule TLR4 antagonists, FP7 and FP12, were tested in human lung (MRC5) and intestinal (CCD-18Co) fibroblast models activated by TGF-β1. Both pre- and post-treatment with these compounds significantly reduced fibroblast proliferation, profibrotic gene expression (fibronectin, collagen I, α-smooth muscle actin), and ECM protein accumulation without inducing cytotoxicity. Imaging analyses confirmed decreased TLR4 and ECM marker expression. Similar antifibrotic effects were observed under pro-inflammatory stimulation of intestinal fibroblasts utilizing three distinct models: the CCD18-Co cell line, primary human colon fibroblasts, and, in a preliminary experiment, an ex vivo human specimen intestinal model, demonstrating broad potential against fibrosis in multiple organ contexts. These results confirm that pharmacological inhibition of TLR4 by small molecule antagonists suppresses fibroblast activation and ECM production, positioning FP7 and FP12 as promising antifibrotic agents. In parallel, the immunostimulatory potential of new synthetic glucosamine-based TLR4 agonists was explored for use as vaccine adjuvants. These agonists were characterized in vitro for selectivity, cytokine induction, and immune cell activation using engineered human cell lines and macrophage models. Compared to the clinically approved adjuvant monophosphoryl lipid A (MPLA), the new compounds showed potent, TLR4-dependent immunostimulation with enhanced stability and tunability, as well as favorable safety profiles. An innovative FTIR screening method helped to identify effective TLR4 activators, supporting the development of novel vaccine adjuvants capable of enhancing both innate and adaptive immunity. Overall, this thesis provides a comprehensive investigation into targeting TLR4 signaling with small molecules for two distinct applications: antifibrotic therapy and vaccine enhancement. By demonstrating the ability of selective TLR4 antagonists to inhibit fibrosis and synthetic TLR4 agonists to boost immunogenicity, this work advances our understanding of TLR4 as a versatile therapeutic target. These findings offer important translational implications, suggesting new pharmacological strategies for managing fibrotic diseases and improving vaccine efficacy.