Successful therapy of lethal murine visceral leishmaniasis with cystatin involves up-regulation of nitric oxide and a favorable T cell response

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4+ T cell differentia tion. A 6-fold increase in NO production was observed with 0.5 muM chicken cystatin, a natural cysteine protease inhibitor, in IFN-gamma -activated ma crophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, comple te elimination of spleen parasite burden was achieved;ed by cystatin in syn ergistic activation with a suboptimal dose of IFN-gamma, In contrast to the case with promastigotes, cystatin and IFN-gamma inhibited the growth of am astigotes in macrophages. Although In vitro cystatin treatment of macrophag es did not induce any NO generation, significantly enhanced amounts of NO w ere generated by macrophages of cystatin-treated animals, Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, an d the increased NO production was paralleled by a concomitant increase in a ntileishmanial activity. Moreover, splenocyte supernatants treated with ant i-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation, where as i.v. administration of these anticytokine Abs along with combined therap y reversed protection against infection, mRNA expression and flow cytometri c analysis of infected spleen cells suggested that cystatin and IFN-gamma t reatment, in addition to greatly reducing parasite numbers, resulted in red uced levels of IL-4 but increased levels of IL-12 and inducible NO synthase , Not only was this treatment curative when administered 15 days postinfect ion, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4(+) differentiation from Th2 to Th1, indicative of long-term resistan ce.