P32 Toll-like receptor (TLR)5 mediates deleterious effects of lipid injury and fibrogenic responses in metabolic dysfunction-associated steatohepatitis (MASH) and clinical outcomes

<h3>Background and Aims</h3> Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive inflammatory liver condition characterised by hepatocyte lipotoxicity, infiltration of inflammatory and immune cells and stellate cell activation leading to fibrosis. The gastrointestinal tract is an important source of inflammatory mediators which activate hepatic Toll-like receptor (TLR) signalling. Stool from patients with MASH have increased proportions of flagellated bacteria (Firmicutes and Proteobacteria phyla). Flagellin is a direct agonist of TLR5 and we test the hypothesis that activation of TLR5 mediates the pathology observed in MASH and fibrosis. <h3>Method</h3> Plasma flagellin and stool Flic gene load (shotgun metagenomics sequencing) were measured in samples from 69 MASLD patients and 11 controls. Lipotoxicity was modelled in vitro using oleic (1mM) and palmitic (0.5mM) acid in primary hepatocytes and stellate (LX2) cells. Hepatic TLR5 mRNA expression and linked healthcare outcomes were extracted from SteatoSITE 1 (n=668 MASLD, 39 controls). <h3>Results</h3> Compared to controls, plasma flagellin is increased in advanced MASH fibrosis (F3–4) but not earlier stages of disease ((538.3v 745.8pg/ml,p&lt;0.01) or in hepatitis B virus infection) which normalises in samples taken median 83 days following bariatric surgery (n=20, p&lt;0.01). Stool Flic gene expression and hepatic TLR5 (but not TLR2 or TLR4) expression are increased in MASH, along with markers of intestinal permeability (FABP2, D-lactate). In vitro, TLR5 inhibition does not alter lipid accumulation (BODIPY) or beta oxidation, but attenuates lipotoxicity-mediated IL8 protein expression (p&lt;0.01, vs control) in primary hepatocytes. TLR5 is not expressed in stellate cells and flagellin does not activate LX2 cells (nor do toxic lipids). However lipid-injured hepatocyte-conditioned media induces 1.4-fold increase in TGFb1 gene expression in LX2 cells compared to control (p=0.01), and Th1 cell differentiation; both of which are reduced through TLR5 inhibition on the hepatocytes prior to media transfer (1.6-fold,p&lt;0.01). High hepatic TLR5 expression is associated with greater hepatic decompensation events and all-cause mortality (figure 1, Kaplan-Meier time-to-event analysis with log-rank test, P&lt;0.01 for both). <h3>Conclusion</h3> TLR5 mediates key pathology in the progression of human MASH and fibrosis, reverses following bariatric surgery and is associated with mechanisms of lipid-mediated inflammation and stellate cell activation. This pathway has potential as a novel therapeutic target in MASH fibrosis.