Bothrops jararaca Snake Venom Inflammation Induced in Human Whole Blood: Role of the Complement System

Abstract

The clinical manifestations of envenomation by Bothrops species are complex and characterized by prominent local effects that can progress to tissue loss, physical disability, or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process originating from tissues damaged by the bothropic venom. Considering the important role that the complement system plays in the inflammatory response, in this study, we analyzed the action of Bothrops jararaca snake venom on the complement system and cell surface receptors involved in innate immunity using an ex vivo human whole blood model. B. jararaca venom was able to induce activation of the complement system in the human whole blood model and promoted a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca reduced the expression of CD11b, CD14 and C5aR1. Inhibition of the C3 component by Cp40, an inhibitor of C3, resulted in a reduction of C3a/C3a-desArg, C5a/C5a-desArg and sTCC to basal levels in samples stimulated with the venom. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8/CXCL8, MCP-1/CCL2 and MIG/CXCL9 in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8/CXCL8 and MCP-1/CCL2. C5aR1 inhibition with PMX205 also promoted a reduction of TNF-α and IL-8/CXCL8 to basal levels in the samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system promoted by the venom of the snake B. jararaca in the human whole blood model significantly contributes to the inflammatory process. The control of several inflammatory parameters using Cp40, an inhibitor of the C3 component, and PMX205, a C5aR1 antagonist, indicates that complement inhibition may represent a potential therapeutic tool in B. jararaca envenoming.

Keywords: Bothrops jararaca; complement system and inhibitors; human whole blood; inflammation; snake venom.

Figure 1

C3a/C3a-desArg, C4a/C4a-desArg and C5a/C5adesArg levels in human plasma after treatment with B. jararaca venom. Human blood samples containing the peptide GPRP (8 mg/mL) were incubated with increasing concentrations of B. jararaca venom or sterile saline solution for 30, 60 and 120 minutes at 37°C. After plasma collection and dilution, the production of anaphylatoxins C3a/C3a-desArg (1:5000), C4a/C4a-desArg (1:5000) and C5a/C5a-desArg (1:1000) was evaluated by cytometric bead array (CBA). The results are presented as the mean ± SEM from three independent tests. ns, not significant; *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001.

Figure 2

SC5b-9 levels in human plasma after exposure to B. jararaca venom. Human blood samples containing the peptide GPRP (8 mg/mL) were incubated with increasing concentrations of B. jararaca venom or sterile saline solution for 30, 60 and 120 minutes at 37°C. After plasma collection and dilution (1:40), the presence of the soluble complement terminal complex (sTCC, SC5b-9) was analyzed by ELISA. The results are represented as the mean ± SEM of duplicates from three independent experiments. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001.

Figure 3

TNF-α levels in human plasma after treatment with B. jararaca venom. Human blood samples containing GPRP (8 mg/mL) were incubated with increasing concentrations of B. jararaca venom or sterile saline solution for 30, 60 and 120 minutes at 37°C. After plasma collection and dilution (1:2), the presence of TNF-α was evaluated by cytometric bead array (CBA). The results are represented as the mean ± SEM of duplicates from three independent tests. ns, not significant; **p ≤ 0.005.

Figure 4

Chemokine levels in human plasma after treatment with B. jararaca venom. Human blood samples containing GPRP (8 mg/mL) were incubated with increasing concentrations of B. jararaca venom or sterile saline solution for 30, 60 and 120 minutes at 37°C. After plasma collection and dilution (1:2), the presence of chemokines was evaluated by cytometric bead array (CBA). The results are represented as the mean ± SEM of duplicates from three independent tests. ns, not significant; *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001.

Figure 5

Expression of surface markers in monocytes and granulocytes after treatment with B. jararaca venom. Human blood samples containing GPRP (8 mg/mL) were treated with B. jararaca venom (50 μg/mL) or sterile saline solution (negative control) for 60 minutes at 37°C. After incubation, cells were analyzed for the expression of CD11b, CD14 and C5aR. The results are expressed as MFI ± SEM of duplicates from three independent experiments. ns, not significant; *p ≤ 0.05, **p ≤ 0.005.

Figure 6

Production of anaphylatoxins in human blood stimulated with B. jararaca venom in the presence of Cp40. Human blood samples containing GPRP (8 mg/mL) were preincubated with cp40 inhibitor (20 μM) and then incubated with B. jararaca venom (50.0 μg/mL) or sterile saline solution for 60 minutes at 37°C. After plasma collection and dilution, the production of anaphylatoxins C3a/C3a-desArg (1:5000), C4a/C4a-desArg (1:5000) and C5a/C5a-desArg (1:1000) was evaluated by cytometric bead array (CBA). The results are presented as the mean ± SEM from three independent experiments. ns, not significant; *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001.

Figure 7

Levels of TNF-α and chemokines in human blood stimulated by B. jararaca venom in the presence of Cp40. Human blood samples containing GPRP (8 mg/mL) were preincubated with Cp40 inhibitor (20 μM) and then incubated with B. jararaca venom (50.0 μg/mL) or sterile saline solution for 60 minutes at 37°C. After plasma collection and dilution (1:2), the presence of TNF-α and the chemokines IL-8, MCP-1 and MIG was evaluated by the Cytometric Bead Array (CBA). The results are presented as the mean ± SEM from three independent experiments. ns, not significant; *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001.

Figure 8

Expression of surface markers in monocytes and granulocytes after treatment with B. jararaca venom in the presence of C-inhibitors. Human blood samples containing GPRP (8 mg/mL) were pre incubated with Cp40 inhibitor (20 μM) or with PMX205 (10 μM) and then incubated with sterile saline (Cp40 vehicle), sterile saline + 5% glucose solution (PMX205 vehicle) or B. jararaca venom (50.0 μg/mL) for 60 minutes at 37°C. After incubation, cells were analyzed for the expression of CD11b, CD14 and C5aR. The results are expressed as MFI ± SEM of duplicates from three independent experiments. ns, not significant; *p ≤ 0.05.

Figure 9

TNF-α and chemokine levels in human blood stimulated by B. jararaca venom in the presence of PMX205. Human blood samples containing GPRP (8 mg/mL) were preincubated with PMX205 (10 μM) and then incubated with B. jararaca venom (50.0 μg/mL) or sterile saline + 5% glucose solution for 60 minutes at 37°C. After plasma collection and dilution (1:2), the presence of TNF-α and chemokines was evaluated by the Cytometric Bead Array (CBA). The results are presented as the mean ± SD from three independent experiments. ns, not significant; **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001.