TLR4: A Potential Therapeutic Target in the Experimental Pathogenesis of Hemolytic-Uremic Syndrome

Abstract

Childhood infections by Shiga toxin (Stx)-producing Escherichia coli (STEC) cause bloody diarrhea and the life-threatening hemolytic-uremic syndrome (HUS). Stx2, produced by STEC in the gut, is released into the bloodstream and interacts with circulating cells (monocytes, neutrophils, and platelets) through two different receptors: Gb3Cer (globotriaosylceramide) and Toll-like receptor 4 (TLR4). Consequently, blood cells form leukocyte/platelet aggregates and release pathogenic Stx2-containing extracellular vesicles (EVs) that, by damaging the kidney, induce the transition from bloody diarrhea to HUS. Human blood was incubated with Stx2 to induce the formation of neutrophil/platelet and platelet-only aggregates (observed in May–Grünwald Giemsa-stained blood smears) and pathogenic blood cell–derived EVs (isolated by size-exclusion chromatography or differential centrifugation) toxic to Vero cells (assessed by a viability assay). In the presence of a monoclonal antibody against TLR4 (anti-TLR4), the formation of neutrophil/platelet and platelet-only aggregates was greatly reduced compared to treatment with Stx2 alone. Moreover, anti-TLR4 significantly decreased the total mass of pathogenic blood cell–derived EVs produced by Stx2 (detected by nanoparticle tracking analysis), which proved less toxic to Vero cells than those produced by Stx2 alone. The impairment of the Stx2-TLR4 axis obtained during the experimental pathogenesis of HUS provides proof-of-concept evidence supporting TLR4 as a potential therapeutic target.