Shiga Toxin 2a Induces NETosis via NOX-Dependent Pathway

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.

Keywords: NADPH-oxidase-dependent pathway; STEC-HUS; hemolytic uremic syndrome; neutrophil extracellular traps; shiga toxin.

Figure 1

Stx2a-treated PMNs from healthy controls show expression of CD11b, an activation marker. Healthy control neutrophils were incubated with media (−ve control) or different concentrations of Stx2a over a period of 8 h. Immunostained neutrophils treated with Stx2a showed upregulated expression of CD11b in a concentration-dependent manner, and thus indicate activation of neutrophils. Images were captured with use of airy scan confocal microscopy at 63× magnification under oil immersion (n ≥ 3 and scale bar 20 µm).

Figure 2

Dose-dependent NETs release response in neutrophils treated with Stx2a. Healthy control neutrophils were treated with different dosages of Stx2a, PMA, media (−ve control) and Triton (+ve control) and fluorescence reading was recorded up to 480 min. A dose-dependent effect was seen between Stx2a concentrations 0.01 µg/mL and 0.1 µg/mL, but not between 0.1 and 1.0 µg/mL of Stx2a. All significant differences were noted in conditions including 1.0 µg/mL, 0.1 µg/mL and PMA to media (−ve control) at indicated time points by a Mixed Models statistical test (* p < 0.05, n = 5).

Figure 3

Immunostained images confirmed the release of NETs in Stx2a treated PMNs. Healthy control neutrophils were treated with media (−ve control) and different dosages of Stx2a. Cells were fixed and immunostained for citrullinated histone 3 (CitH3), myeloperoxidase (MPO) and DNA after 4, 6 and 8 h. NETs are visible as web-like structures positive for DNA (blue) decorated with myeloperoxidase (red). Staining of CitH3 (green) is not reported, indicating that Stx2a induces the Nox-dependent NETosis pathway (63× Magnification, n = 3 and scale bar 20 µm). For positive control and quantification, see Supplementary Figure S1.

Figure 4

Inhibitors data confirmed that Stx2a induces NETosis via the NOX-dependent pathway. Neutrophils were treated with media (−ve cotrol), Stx, and PMA with and without different inhibitors (DPI, FR, NAC and SB20190) and a NETosis kinetics assay was performed. (a–e) Significant inhibition of NETs was found in neutrophils treated with Stx2a-at concentrations of 0.1 and 1.0 µg/mL after 4 h incubation when neutrophils were preincubated with FR, NAC and SB20190 (b,c). However, significant inhibition of DNA release was found for all Stx2a concentrations (0.01, 0.1 and 1.0 µg/mL) when neutrophils were preincubated with the NOX-specific inhibitor DPI (b–d). This strongly suggests a pivotal role of NOX in Stx2a-mediated NETosis, when considering a similar response of PMA-treated neutrophils (e) (* p < 0.05 compared to their respective control, 2-Way ANOVA with Bonferroni posttest, n = 4). For SytoxGreen plate images, and kinetics data, see supplementary Figures S2 and S3.

Figure 5

Stx2a induces NOX-ROS generation. Release of NOX-ROS and mitochondrial-ROS was measured by using DHR123, and MitoSox respectively. PMA and ionomycin were used as +ve control. (A) Data shows the involvement of NOX-ROS in Stx2a treated cells but not of mitochondrial ROS, there only +ve control (ionomycin) showed mitochondrial ROS generation (B) NOX-ROS release was measured along with DPI—a NOX-specific inhibitor, or NAC—a ROS scavenger. Untreated neutrophils (a) treated with 1.0–0.01 µg/mL of Stx2a (b–d) or 25 nM of PMA (e) with inhibitors over a time period of 90 min showed NOX-ROS generation. Significant differences were determined by a Mixed Models statistical test (p < 0.05). Significant differences were noted in conditions with their media control at respective time points (* p < 0.05 Stx2a compared to inhibitors DPI and NAC at respective time points, n = 4).

Figure 6

Spontaneous NETosis of neutrophils derived from patients with STEC-HUS. Immunostained images after 4 h of incubation show spontaneous release of NETs by neutrophils derived from patients in acute and recovery phase of disease, while this effect is not seen in healthy control neutrophils. Web-like structures representing NETs are positively stained for DNA (blue) and MPO (red). 63× magnification, n = 3 and scale bar 20 µm.

Figure 7

Release of DNA from neutrophils of STEC-HUS patients. Neutrophils derived from healthy donors and STEC-HUS patients in the acute phase and recovery phase of disease were incubated with (a) PMA, (b) LPS, (c) A23187 and (d) Ionomycin for 4 h to stimulate NETs release. Data show less release of NETs by neutrophils in the acute phase of disease in both the NOX-dependent pathway and also in the NOX-independent pathway but with lesser extent. (* p ≤ 0.05, ** p ≤ 0.01, n > 3, Friedman test and Wilcoxon test were applied).

Figure 8

Release of ROS from neutrophils of STEC-HUS patients. Neutrophils derived from healthy donors and STEC-HUS patients in the acute phase and recovery phase of disease were incubated with (a) PMA, (b) LPS, (c) Ionomycin, (d) A23187 for 4 h to estimate ROS generated by NOX-dependent and -independent pathways. Data show less generation of ROS by neutrophils in the acute phase of disease when the NOX-dependent pathway is activated. The respective significant comparisons are indicated by horizontal bar with (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, n = 3, Friedman test and Wilcoxon test were applied).