Pathogenic Leptospira spp Induce Higher Superoxide Anion Production by HL 60-derived Neutrophils

Abstract

The superoxide anion (O· 2 -) is the first free radical produced in phagocytes which enables pathogen killing and also generates other microbicidal reactive oxygen species (ROS).The induction of high O· 2 - production by phagocytes as a virulence factor for pathogenic Leptospira is not known. The objective of this study was to assess the induced O· 2 - production by human neutrophils exposed Leptospira and compare the differences in the level of O· 2 - produced by HL-60 derived neutrophils interacted with pathogenic and saprophytic Leptospira spp. HL-60 cells were treated with dimethyl sulfoxide to differentiate in to mature neutrophils. Saprophytic Leptospira biflexa serovar Patoc and pathogenic L. Interrogans serovar Pyrogenes were grown in Ellinghausen-McCullough-Johnson-Harris media containing 10% heat inactivated rabbit sera. Intracellular O· 2 - production of neutrophils interacted with bacteria in the presence of sera from leptospirosis patients (n=30/group of severe and mild leptospirosis patients) and healthy subjects (n=20) was assessed using quantitative nitroblue tetrazolium assay. Both anti-leptospiral IgM and IgG levels were measured in test sera prior to the assay. In the presence of normal and also patients’ sera, neutrophils interacted with L. Interrogans Pyrogenes produced significantly high O· 2 - levels than that of L. biflexa (p<0.05). When Leptospira density was varied for neutrophil-bacteria interactions, low Leptospira densities induced higher O· 2 - productions compared to the high concentrations (p<0.03). Moreover, analysis of O· 2 - production by neutrophils in the presence of patients’ sera showed that, opsonization of both saprophytic and pathogenic Leptospira by IgM or IgG had not induced a significant difference in the level of O· 2 - produced (p = 0.012). These results suggest that, the increased O· 2 - production induced by L. Interrogans Pyrogenes could be a virulent factor in pathogenic Leptospira spp which may contribute to the pathogenesis of severe leptospirosis.