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

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.