“Increased NADPH oxidase subunit gp91phox and TLR2 expressions are associated with reactive oxygen species production induced by cruzipain”.

GUIÑAZU N, BECERRA C, PELLEGRINI A, CARRERA-SILVA EA, CANO R, ALBESA I, GEA S.

Rio de Janeiro, Brasil

Congreso; 13th Internacional Congress of Immunology.; 2007

Recently, we demonstrated that the immunization of C57BL/6 mice with cruzipain (Cz), a Trypanosoma cruzi antigen, generated a pro-inflammatory immune response with nitric oxide production which participated in the control of parasite replication. It have been postulated that reactive oxygen species (ROS) play an indispensable role in controlling the growth of pathogens. However, the identification of the T. cruzi molecular components able to stimulate ROS production has not been fully explored. Here, we investigated the ability of Cz to modify the oxidative metabolism of spleen cells from Cz immunized or non immunized C57BL/6 mice, and in Raw 264.7 cells. Using luminol-enhanced chemiluminescence, we determined that in vitro treatment with Cz increased the oxidative burst in all these cell types. To elucidate whether O2 -, H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.antigen, generated a pro-inflammatory immune response with nitric oxide production which participated in the control of parasite replication. It have been postulated that reactive oxygen species (ROS) play an indispensable role in controlling the growth of pathogens. However, the identification of the T. cruzi molecular components able to stimulate ROS production has not been fully explored. Here, we investigated the ability of Cz to modify the oxidative metabolism of spleen cells from Cz immunized or non immunized C57BL/6 mice, and in Raw 264.7 cells. Using luminol-enhanced chemiluminescence, we determined that in vitro treatment with Cz increased the oxidative burst in all these cell types. To elucidate whether O2 -, H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.T. cruzi molecular components able to stimulate ROS production has not been fully explored. Here, we investigated the ability of Cz to modify the oxidative metabolism of spleen cells from Cz immunized or non immunized C57BL/6 mice, and in Raw 264.7 cells. Using luminol-enhanced chemiluminescence, we determined that in vitro treatment with Cz increased the oxidative burst in all these cell types. To elucidate whether O2 -, H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.in vitro treatment with Cz increased the oxidative burst in all these cell types. To elucidate whether O2 -, H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.treatment with Cz increased the oxidative burst in all these cell types. To elucidate whether O2 -, H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.2 -, H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells., H2O2 or ONOO- was predominantly produced, the cells were co-cultured with superoxide dismutase (SOD) or TIRON, catalase and LNMMA respectively. SOD or TIRON significantly diminished ROS production, suggesting that Cz mainly stimulated O2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.2 - generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.generation. Additionally, western blot analysis showed that Cz up-regulated the NADPH oxidase catalytic subunit gp91phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.phox in spleen cells. Taking into account that toll like receptors were reported recently to mediate the oxidative burst, Cz was tested for its ability to modulate TLR2 or TLR4 expression. Along with the enhanced oxidative burst, Cz stimulation also increased TLR2 but not TLR4 protein. Besides, this parasite antigen induced IL6 and IL12 but not TNF�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.�� pro-inflammatory cytokine secretion. Together these results demonstrate that Cz favors ROS production by NADPH oxidase and leads to TLR2 up-regulation on murine cells.