INCREASED LEVELS OF NEUTROPHIL EXTRACELLULAR TRAPS IN HUS PATIENTS

RAMOS, M. VICTORIA; MEJIAS MP; SABBIONE, F; FERNÁNDEZ BRANDO, ROMINA J; SANTIAGO, AP; AMARAL, MM; EXENI R; TREVANI, A; PALERMO, MARINA S

Boston

Congreso; 9th International Symposium On Shiga Toxin (Verocytotoxin) Producing Escherichia coli Infections.; 2015

VTEC

Introduction:HemolyticUremic Syndrome (HUS), is a vascular disease caused by Shiga toxin (Stx) butalso inflammatory response mediatedby neutrophils (PMN) is essential to HUS evolution. PMN can release "neutrophilextracellular traps" (NETs) composed of DNA, histones and proteins such aselastase and myeloperoxidase. Since NETs are involved in several inflammatorydiseases, the aim of this work was toinvestigate the presence  of NETs in HUSpatients. Methods: Peripheral blood from acute HUS and healthy children(HC)(n=15) was collected, plasmas were separated and PMN were isolated bygradients. PMN were incubated with medium or phorbolmyristateacetate (PMA) or monosodium-uratecrystals (MSU), as inducers of NETosis. After 4h, circulating free-DNA (cf-DNA)concentration and elastase activity weremeasured in the supernatants by fluorometry and spectrophotometry respectively.Colocalization of DNA and myeloperoxidase in plasma was analyzed by immunofluorescenceand confocal microscopy. To evaluate the effect of NETs on endothelium, HumanGlomerular Endothelial Cells (HGEC) were incubated during 24h with NETs, then cell viability was evaluated by spectrophotometry andcytokine levels in the supernatants were evaluated by ELISA.  Results and Discussion: There was an increase in cf-DNA levels in plasma fromHUS compared to HC (HC=5,1±0,5;HUS=10±0,5*, Mean±SEM, ug/ml, n=15, *p<0,05). Moreover, cf-DNA colocalizedwith myeloperoxidase in HUS patients´ plasmas. Stimulated PMN from bothclinical groups,  showed similar capacityto produce NETs, evaluated as DNA and elastase levels. Cell viability of HGECstimulated with NETs was not affected but there was an increase in secretion of inflammatory-cytokines IL-6 and IL-8 (Mean±SEM (IL-8=Basal:24.9±1.9, NETs: 40.1±4.5*;IL-6=Basal:  8.9±1.0, NETs:14.9±1.2*), ng/mL; n=5, *p<0,05).Implications: The increased levels of NETs in plasma duringHUS, could be consequence of the direct Stx2 capacity to induce NETosis of PMN,(but also of other inflammatory mediators). Moreover, these structures can activate endothelium,inducing the  secretion of inflammatorycytokines. All these together, suggests that NETs could be implicated inamplification of inflammatory response.