Crotalus durissus collilineatus Venom Induces TNF- α and IL-10 Production in Human Peripheral Blood Mononuclear Cells

Abstract

Snake venom has been the subject of numerous studies in an attempt to find properties and biological effects that may be beneficial to man. In this study we evaluated in vitro the effects of Crotalus durissus terrificus (Cdt) and Crotalus durissus collilineatus (Cdc) venom in human peripheral blood mononuclear cells (PBMCs). At 24 h, a significant decrease of viable cells was observed in cells stimulated with the Cdc venom at 0.0005 mg/mL and 0.005 mg/mL compared to the negative control. At 48 h, a significant decrease of viable cells was observed only in cells stimulated with Cdc venom at 0.005 mg/mL. A significant increase of TNF- α and IL-10 was detected 48 hours after culture of PBMC with Cdc, but not with Cdt venom. The expression of CD69 and PD1 (programmed death-1), activation and regulatory cell markers, on CD8+ and CD8- T cells did not change in the presence of Cdt and Cdc venom. Our results suggest the presence of proinflammatory and anti-inflammatory components in the Cdc venom. Further analysis should be done to identify those Cdc venom components as it has been done for the Cdt venom by other authors, indicating that modulatory components are found in the venom of different species of Crotalus snakes.

Figure 1

PBMC counts after 24, 48, and 72 hours of culture in the presence and absence of Cdt and Cdc venom. An initial amount of 2 × 10⁵ cells/mL was stimulated with Cdt venom and Cdc venom in concentrations of 0.0005 μg/mL and 0.005 μg/mL and only with RPMI (NS). (a) PBMC counts in 24 hours with Cdt venom; (b) PBMC counts in 48 hours with Cdt venom; (c) PBMC counts in 72 hours with Cdt venom. (d) PBMC counts in 24 hours with Cdc venom; (e) PBMC counts in 48 hours with Cdc venom; (f) PBMC counts in 72 hours with Cdc venom. The analysis was done by Mann-Whitney test and the horizontal bars show the median.*P < 0.05 when compared with the corresponding negative control. Cdt: Crotalus durissus terrificus venom; Cdc: Crotalus durissus collilineatus venom.

Figure 2

Concentration of cytokines produced by PBMCs after 48 hours of culture in the presence and absence of Cdt and Cdc venom. Concentrations of (a) IFN-γ, (b) TNF-α, and (c) IL-10. 2 × 10⁵ cells/mL were stimulated with different concentrations of the venom and the culture supernatants were collected after 48 hours. Cytokines in the supernatants were measured by Cytometric Bead Array. The analysis was done by Mann-Whitney test and the horizontal bars show the median.*P < 0.05 when compared with the corresponding negative control. Cdt: Crotalus durissus terrificus venom; Cdc: Crotalus durissus collilineatus venom; NS: nonstimulated cells.

Figure 3

Expression of activation (CD69) and regulation (PD1) markers. PBMCs were cultured in presence of Cdt and Cdc venom. After 24 h, cells were stained with antibodies for CD8, PD1, and CD69 and analyzed by flow cytometry. A gate was done in the region of lymphocytes and those cells were gated as CD8+ and CD8− subsets and the expression of PD1 and CD69 was analyzed inside of these subsets. (a) CD69 expression on CD8+ T cells. (b) PD1 expression on CD8+ T cells. (c) CD69 expression on CD8− T cells. (d) PD1 expression on CD8− T cells. The analysis was done using Mann-Whitney test and the horizontal bars show the median. Cdt: Crotalus durissus terrificus venom; Cdc: Crotalus durissus collilineatus venom; NS: nonstimulated cells.