Erythrocyte TLR9 is upregulated in metabolic associated fatty liver disease, and is linked to an inflammatory immunometabolic signature.

Metabolic associated fatty liver disease (MAFLD) consists of lipid accumulation in the liver. Lipotoxicity, supported by aberrant amino acid metabolism, induces TLR9 upregulation, and activation, driving inflammation. Relatively, erythrocyte TLR9 activation leads to membrane rearrangement, surface CD47 loss, and pro-inflammatory erythrophagocytosis. Erythrocyte surface protein loss is accompanied by chemokine release. In addition, CD47 binds circulating TSP-1, a molecule controlling arginine and glutamine metabolism, along with metabolic inflammation. Based on these, we speculated that in MAFLD, lipotoxicity would drive erythrocyte TLR9 upregulation and activation, leading to immunometabolic remodeling. Twenty-four patients (15 men and 9 women) with MAFLD and 9 healthy controls (4 men and 5 women) were enrolled. Erythrocytes were isolated from EDTA-containing blood. Protein levels were measured in erythrocyte lysates (triton X-100 0.01% v/v) or plasma with enzyme-linked immunosorbent assays, whereas lipids and enzyme activities were measured in erythrocyte hemoglobin-free membranes by a semi-quantitative thin layer chromatography and assay kits, respectively. The levels of TLR9 were increased (p=0.002) and positively correlated with sphingosine levels, albeit not statistically significantly (p=0.060). The erythrocyte membrane PC/PE ratio was decreased (p=0.002) and inversely correlated to TLR9 levels. Erythrocyte TLR9 levels correlated inversely with CD47, and positively with MCP-1 release. TSP-1 was decreased in MAFLD erythrocytes (p=0.0017) and correlated positively with CD47 and negatively with TLR9 levels. In vitro, erythrocytes of MAFLD patients bound less TSP-1 molecules. Erythrocytes of MAFLD patients also exhibit decreased arginase-1 protein (p=0.009) and activity (p=0.042). Glutaminase activity was increased, albeit to not statistically significantly different levels (p=0.25). The levels of CD35 were not different (p=0.65), excluding the role of erythrocyte aging for explaining these events. In MAFLD patients, erythrocyte TLR9 is upregulated and is associated with erythrocyte sphingolipid and glycerophospholipid perturbation, CD47 loss, MCP1 release, reduced TSP-1 scavenging, decreased arginase and increased glutaminase.