Neutrophil recruitment during Leishmania infection: The role of sand fly salivary proteins

Neutrophils are the first cells recruited to the site of an infected sand fly bite and interact with Leishmania parasites. It has been proposed that the recruitment of neutrophils to the site of the bite increases the ability of Leishmania to establish an infection. Here we investigate the effect of sand fly salivary proteins on neutrophil recruitment. We purified bone marrow neutrophils from C57BL/6 mice or from peripheral blood of healthy human donors. Chemotaxis driven by sand fly saliva of vectors of visceral ( Lutzomyia longipalpis ) or cutaneous ( Phlebotomus duboscqi ) disease was measured by modified Boyden chamber assay and by imaging with the EZ-TAXIScan assay. Neutrophils migrated towards saliva glands of the vectors P. duboscqi and L. longipalpis , in a dose-dependent manner. Proteinase K treatment of saliva completely abrogated neutrophil recruitment, suggesting a protein as the chemotactic factor. To unearth the identity of the chemotactic factor, we have generated plasmids coding for the 20 most abundant salivary proteins from P. duboscqi and injected them in the ears of C57BL/6 mice. Neutrophil recruitment was analyzed by flow cytometry staining. We have identified 3 different salivary protein-coding plasmids with neutrophil recruitment activity. Finally, we have produced these 3 recombinant proteins in the HEK293 cell line system and we observed that a family of 40kDa salivary proteins acts as chemokines for neutrophils. We plan to validate the role of the salivary chemotactic factor in vivo and test if its neutralization can alter Leishmania infection. We put forward the hypothesis that blockage of this protein will disrupt neutrophil migration early in the Leishmania infection possibly altering the disease outcome.