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Human Glomerular Endothelial Cells Treated With Shiga Toxin Type 2 Activate γδ T Lymphocytes.

ABSTRACT: The hemolytic uremic syndrome associated with diarrhea, a consequence of Shiga toxin (Stx)-producing Escherichia coli infection, is a common cause of pediatric acute renal failure in Argentina. Stx type 2a (Stx2a) causes direct damage to renal cells and induces local inflammatory responses that involve secretion of inflammatory mediators and the recruitment of innate immune cells. γδ T cells constitute a subset of T lymphocytes, which act as early sensors of cellular stress and infection. They can exert cytotoxicity against infected and transformed cells, and produce cytokines and chemokines. In this study, we investigated the activation of human peripheral γδ T cells in response to the incubation with Stx2a-stimulated human glomerular endothelial cells (HGEC) or their conditioned medium, by analyzing in γδ T lymphocytes, the expression of CD69, CD107a, and perforin, and the production of TNF-α and IFN-γ. In addition, we evaluated by confocal microscopy the contact between γδ T cells and HGEC. This analysis showed an augmentation in cellular interactions in the presence of Stx2a-stimulated HGEC compared to untreated HGEC. Furthermore, we observed an increase in cytokine production and CD107a expression, together with a decrease in intracellular perforin when γδ T cells were incubated with Stx2a-treated HGEC or their conditioned medium. Interestingly, the blocking of TNF-α by Etanercept reversed the changes in the parameters measured in γδ T cells incubated with Stx2a-treated HGEC supernatants. Altogether, our results suggest that soluble factors released by Stx2a-stimulated HGEC modulate the activation of γδ T cells, being TNF-α a key player during this process.

SUBMITTER: Rosso DA

PROVIDER: S-EPMC8656354 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN-Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN-Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN-Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS.

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Human Glomerular Endothelial Cells Treated With Shiga Toxin Type 2 Activate γδ T Lymphocytes.

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