Sacerdoti, F. | Search bioRxiv

bioRxiv2022

Detection and characterization of circulating microvesicles containing Shiga toxin type 2 (Stx2) in a rat model of Hemolytic Uremic Syndrome

Shiga toxin (Stx) producing Escherichia coli (STEC) are foodborne pathogens that release Stx and may develop Hemolytic Uremic Syndrome (HUS). Stx causes endothelial cell damage and leads to platelets deposition and thrombi formation within the microvasculature. It has been described that Stx activates blood cells and induces the shedding of proinflammatory and prothrombotic microvesicles (MVs) containing the toxin. In this sense, it has been postulated that MVs containing Stx2 (MVs-Stx2+) can contribute to the physiopathology of HUS, allowing Stx to reach the target organs and evading the immune system. In this work, we propose that circulating MVs-Stx2+ can be a potential biomarker for the diagnosis and prognosis of STEC infections and HUS progression. In this regard, we developed a rat HUS model by the intreperitoneal injection of a sublethal dose of Stx2 and observed: decrease in body weight, increase of creatinine and urea levels, decrease of creatinine clearance and histological renal damages. After characterization of renal damages we investigated circulating total MVs and MVs-Stx2+ by flow cytometry at different times after Stx2 injection. Additionally, we evaluated the correlation of biochemical parameters such as creatinine and urea in plasma with MVs-Stx2+. As a result, we found a significant circulation of Mvs-Stx2+ at 96 hours after Stx2 injection, nevertheless no correlation with creatinine and urea plasma levels were detected. Our results suggest that MVs-Stx2+ may be an additional biomarker for the characterization and diagnosis of HUS progression. Further analysis is required in order to validate MVs-Stx2+ as biomarker of the disease.

pathology↗

bioRxiv2021

Effects of bacterial lipopolysaccharide and Shiga Toxin on induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells

BackgroundMesenchymal Stem Cells can be activated and respond to different bacterial toxins. Lipopolysaccharides (LPS) and Shiga Toxin (Stx) are the two main bacterial toxins present in Hemolytic Uremic Syndrome (HUS) that cause endothelial damage. In this work we aimed to study the response of iPSC-MSC to LPS and/or Stx and its effect on the restoration of injured endothelial cells. MethodsiPSC-MSC were used as a source of mesenchymal stem cells (MSC) and Human Microvascular Endothelial Cells-1 (HMEC-1) as a source of endothelial cells. iPSC-MSC were treated or not with LPS and/or Stx. For some experiments, Conditioned Media (CM) were collected from each plate and incubated with an anti-Stx antibody to block the direct effect of Stx, or Polymyxin to block the direct effect of LPS. In CM from both treatments, anti-Stx and Polymyxin were used. Results are expressed as mean {+/-} S.E.M. Significant differences (p<0.05) were identified using one way analysis of variance (ANOVA) and Bonferronis Multiple comparison test. ResultsThe results obtained showed that LPS induced a pro-inflammatory profile on iPSC-MSC, but not Stx, even though they expressed Gb3 receptor. Moreover, LPS induced on iPSC-MSC an increment in migration and adhesion to gelatin substrate. Also, the addition of CM of iPSC-MSC treated with LPS+Stx, decreased the capacity of HMEC-1 to close a wound, and did not favor the formation of new tubes. Proteomic analysis of iPSC-MSC treated with LPS and/or Stx revealed specific protein secretion patterns that support many of the functional results described here. ConclusionsIn conclusion, these results suggest that iPSC-MSC activated by LPS acquired a pro-inflammatory profile that induces migration and adhesion to extracellular matrix proteins (ECM), but the combination LPS+Stx decreased the repair of endothelial damage. The importance of this work is that it provides knowledge to understand the context in which iPSC-MSC could benefit or not the restoration of tissue injury, taking into account that the inflammatory context in response to a particular bacterial toxin is relevant for iPSC-MSC immunomodulation.

immunology↗