Subsequent murine models, using CP-100356 monohydrochloride either heat-killed and live bacteria or LPS, showed evidence that both TLR2 and TLR4 play a role. Leptospiral LPS was shown to be recognized by both TLR2 and TLR4 in murine cells whereas leptospiral lipoproteins were recognized by TLR2 in murine kidney epithelial cells. Mice with combined TLR2/TLR4 deficiency were found to be highly susceptible to lethal leptospirosis. However, these previous studies might not be representative of the immune response to viable serovars in humans. Hence, in the present study we investigated the innate immune response to several viable leptospiral serovars in human blood. Killing assays and stimulation experiments were performed using a human monocytic cell line, human peripheral blood mononuclear cells and human whole blood. Moreover we examined the involvement of TLR2, TLR4 and TLR5 in the cellular responsiveness to viable Leptospira serovars. During the pathophysiology of leptospirosis leptospires are spread through the blood stream. To explore the proinflammatory potency of this compartment in response to virulent and avirulent leptospires we incubated the different leptospiral strains in a minimally altered whole blood assay. Similar observations were obtained in two different blood donors. Both culture-adapted strains were less potent at inducing TNF-a at low bacterial burden compared to the virulent host-adapted counterparts. At high, OSU6162 hydrochloride non-physiological, concentrations all strains displayed the same potency in whole blood. Similar dose-dependent responses were observed for IL-6 release. In efforts to characterize the response in whole blood we inhibited TLR2, TLR4 and TLR5 in physiologically relevant concentrations of the virulent host-adapted serovar Bataviae. This involvement of different TLRs was studied using cell culture grade inhibitory antibodies from InvivoGen shown to block either human TLR2, TLR4 or TLR5 and developed for this purpose by the manufacturer. We confirmed the inhibitory capacity of the antibody preparations in control experiments.