P010 Cytokine milieu and nitrite release during early challenge of human PBMC by Leishmania infantum and Leishmania major

Introduction Although Th1/Th2 paradigma may account for experimental Leishmaniasis pathogenesis in animals, data on cellular immunity mediators in early phase of human Leishmania infections are still limited and controversial. Therefore the aim of this study was to investigate some Th1 and Th2 cytokines, MCP-1, among the monocyte chemokines, and nitrite release from human PBMC stimulated by Leishmania infantum and Leishmania major metacyclic promastigotes (at a MOI of 2:1 and 0.2:1), the infective form of such parasite, in order to mimic the early phase of the natural infection. Methods After 4 and 24 hours (h) of incubation of PBMC cultures stimulated by live metacyclic Leishmania promastigotes, supernatants were obtained and used for simultaneous determination of cytokine/chemokine levels by a cytokine biochip array on the Evidence Investigator analyser (Randox Laboratories Ltd., Crumlin, UK); as well as for quantitative determination of nitrite by Griess reagent. Results were presented as means ± SEM of three independent experiments each carried out in quadruplicate. Statistic analysis was carried out by Student’s t test. A p value  Results During the very early phase (4 h) of L. major challenge TNF α , IL-1 β , IL-6 levels were significantly (p  L. infantum did not cause any TNF α , IL-1 β and IL-6 release. At 24 h promastigote-stimulated TNF α , IL-1 β , IL-6 levels were significantly ( p L. major produced a greater release of these cytokines than the same concentration of L. infantum. Regarding chemokine release an interesting dose-dependent effect was observed for both species and the higher concentration of the promastigotes of L. infantum and L. major stimulated a more elevated release of MCP-1, yet this chemokine levels evaluated at 4 h were not significantly different vs. controls. The nitrite analysis was carried out in order to estimate the release of nitric oxide, whose nitrite is a stable metabolite. At 4 h the nitrite levels were slightly, but significantly ( p L. major and L. infantum did not cause any significant change of the nitrite level vs. control values. Conclusion It appears that the larger amount of parasites may modulate both Th1 and Th2 cytokines evaluated and, more importantly, inhibit the release of nitric oxide, the host molecule that ultimately will kill such protozoa. On the other side promastigotes of L. infantum and L. major stimulated a significant and dose-dependent release of MCP-1. Our data may aid to spread a light on differential mechanisms used by Leishmania species in order to evade the host immune system.