Cellular apoptosis and nitric oxide production in PBMC and spleen from dogs with visceral leishmaniasis.

Nitric oxide (NO) is involved in the death of the Leishmania parasite and regulation of apoptosis. We quantified the frequency of cells producing NO and its levels in the peripheral blood mononuclear cells (PBMC), leukocytes from spleen in Visceral Leishmaniasis (VL) symptomatic dogs and correlated NO levels with apoptosis and parasite load in the spleen. The percentage of NO+ cells and CD14+/NO+ was higher in PBMC and spleen cells in infected dogs than in controls. The levels of NO+ and CD14+/NO+ cells was higher in PBMC, but lower spleen of dogs infected than compared to control. Late apoptosis rates increased in PBMC and spleen of infected dogs compared to controls, and the NO levels and apoptosis not showed correlation. There was a positive correlation between the percentage of cells producing NO in the spleen and parasite load. The NO participates in the immune response in the canine VL, but it is not apoptosis inducer.

PD-1 function in apoptosis of T lymphocytes in canine visceral leishmaniasis.

Dogs infected with Leishmania infantum have a reduced number of T lymphocytes. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family that is expressed by immune cells, and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces the deactivation or apoptosis of T cells. This study aimed to evaluate the expression of PD-1 and its ligands, as well as blocking in the induction of apoptosis in T lymphocytes, TNF-α, IL-4 and nitric oxide production by leucokocytes from PBMC and spleen and the parasite load in dogs with visceral leishmaniasis (VL). Our results showed that the expression of PD1 and its ligands was increased in CD3(+) T cells and CD21(+) B lymphocytes within the peripheral blood and splenic mononuclear cells of dogs with VL. In peripheral blood monocytes, only PD-1 ligands exhibited increased expression; however, in spleen macrophages, increased expression of both PD-1 and its ligands was observed. Levels of apoptosis in peripheral blood and splenic T lymphocytes were higher in dogs with VL compared to healthy dogs. Blocking monoclonal antibodies to PD-1 and its ligands in the culture of mononuclear cells from the peripheral blood and spleen decreased the amount of CD3(+) T lymphocyte apoptosis. The concentration of nitric oxide, TNF-α and IL-4 increased in the culture supernatants of peripheral blood mononuclear cells treated with a blocking monoclonal antibody against PD-1. The TNF-α concentration increased in the culture supernatants of splenic cells following all treatments with antibodies blocking PD-1 and its ligands; however, the amount of IL-4 increased only in the presence of a PD-1 blocking agent. Treatment with a PD-1 blocking monoclonal antibody in the mononuclear peripheral blood of dogs with VL reduced the parasite burden while increased TNF-α. We conclude that in canine visceral leishmaniasis, PD-1 and its ligands are involved in the induction of T lymphocyte apoptosis and in regulating the production of nitric oxide, TNF-α, and IL-4, as well as the parasitic load.