Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: involvement of ERK1/2, NF-kappaB and JAK/STAT pathways.

Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-gamma in inducing ERK1/2 phosphorylation and NF-kappaB DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-kappaB or ERK1/2 pathway blocked the cystatin plus IFN-gamma-inducible NF-kappaB activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-kappaB-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-gamma-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-kappaB, including nuclear translocation of NF-kappaB subunits, IkappaB phosphorylation and IkappaB kinase, are all remarkably enhanced in Leishmania-infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general.