Shiga Toxin 2a Induces NETosis via NOX-Dependent Pathway.

Wouter J C Feitz,Nades Palaniyar,Lambert P Van Den Heuvel,Carolina G Ortiz-Sandoval,Nicole C A J Van De Kar,Christoph Licht,Samuel Suntharalingham,Meraj Khan

doi:10.3390/biomedicines9121807

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is the most common cause of hemolytic uremic syndrome (HUS), one of the main causes of acute kidney injury in children. Stx plays an important role in endothelium damage and pathogenesis of STEC-HUS. However, the effects of Stx on neutrophils and neutrophil extracellular trap (NET) formation are not well understood. In this study, we investigated how Stx2a affects NET formation and NETotic pathways (NADPH or NOX-dependent and -independent) using neutrophils isolated from healthy donors and patients with STEC-HUS, during the acute and recovery phase of the disease. Stx2a dose-dependently induced NETosis in neutrophils isolated from both healthy controls and STEC-HUS patients. NETosis kinetics and mechanistic data with pathway-specific inhibitors including diphenyleneiodonium (DPI)-, ERK-, and P38-inhibitors showed that Stx2a-induced NETosis via the NOX-dependent pathway. Neutrophils from STEC-HUS patients in the acute phase showed less ROS and NETs formation compared to neutrophils of the recovery phase of the disease and in healthy controls. NETs induced by Stx2a may lead to the activation of endothelial cells, which might contribute to the manifestation of thrombotic microangiopathy in STEC-HUS.

Highlights

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure [1]
These results show that Stx2a upregulates CD11b and induces neutrophil extracellular trap (NET) formation in healthy human neutrophils, in a concentration-dependent manner
NETs via the NOX-dependent pathway with the production of reactive oxygen species (ROS), while the NOX-independent pathway does not seem to be involved as indicated by the absence of substantial mitochondrial-specific ROS production and citrullinated H3 formation

Summary

Introduction

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure [1]. HUS predominantly affects children under the age of 5 years [2]. The majority of pediatric cases is triggered by an infection with Shiga toxin (Stx)-producing Escherichia coli (STEC-HUS), typically caused by ingestion of contaminated food [3]. There is no specific treatment for STEC-HUS. Patients are treated with supportive measures including renal replacement therapy, antihypertensive drugs, red blood cell transfusions, and fluid and electrolyte management [4]. A total of 70% of patients recover with time and supportive therapy [5], and mortality of STEC-HUS ranges from 2–5% [3]

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