Inflammasome NLRP3 activation induced by Convulxin, a C-type lectin-like isolated from Crotalus durissus terrificus snake venom

Abstract

Convulxin (CVX), a C-type lectin-like protein isolated from the venom of the snake species, Crotalus durissus terrificus, stimulates platelet aggregation by acting as a collagen receptor agonist for glycoprotein VI found in the platelets. The effect of CVX on platelets has been studied, but its effect on human peripheral blood mononuclear cells (PBMCs) remains unclear. Given the significance of PBMCs in inflammation, this study explored the effect of CVX on PBMCs, specifically regarding NLRP3 inflammasome activation by assessing cell viability, ability to induce cell proliferation, reactive oxygen species (ROS) and nitric oxide production, interleukin (IL)-2 and IL-10 secretion, NLRP3 complex activation, and the role of C-type lectin-like receptors (CTLRs) in these. CVX was not toxic to PBMCs at the investigated concentrations and did not increase PBMC growth or IL-2 release; however, CVX induced IL-10 release and ROS generation via monocyte activation. It also activated the NLRP3 complex, resulting in IL-1β induction. Furthermore, the interaction between CVX and Dectin-2, a CTLR, induced IL-10 production. CVX interaction with CTLR has been demonstrated by laminarin therapy. Because of the involvement of residues near the Dectin-2 carbohydrate-recognition site, the generation of ROS resulted in inflammasome activation and IL-1β secretion. Overall, this work helps elucidate the function of CVX in immune system cells.

Figure 1

Cell viability of PBMCs in the presence of Convulxin (CVX). Human PBMCs isolated from leukocytes from healthy adult blood donors by a density gradient method viability in the absence or presence of inhibitors was measured by MTT metabolization method (A). Isolated 2 × 10⁵ PBMCs were incubated with RPMI (control) or with different concentrations of CVX (0.3 to 20 μg/mL; experimental group) for 1 h (B), or CVX (5 and 10 μg/mL; experimental group) for 12 (C), 24 (D), 48 (E), 72 (F), and 96 h (G). Viability was then assessed by PI and TO method in FACScan. The results were expressed in % and represent the mean ± S.E.M. from 4 to 5 donors.

Figure 2

Cell proliferation of PBMCs in the presence of Convulxin. CFSE labelling of 2 × 10⁵ PBMCs proliferation followed by stimulation with RPMI (negative control) (A), PHA (5 µg/mL; positive control) (B) or CVX (5 and 10 μg/mL) (C,D) for 72 h and were analyzed in FACScan. The results were expressed as counts in histograms and represent the mean ± S.E.M of 3 donors.

Figure 3

Interleukin-2 (IL-2) and Interleukin-10 (IL-10) production in PBMCs in the presence of Convulxin. Cytokines quantification in the supernatant of 2 × 10⁵ PBMCs by EIA followed by stimulation with RPMI (negative control), ConA (5 µg/mL; positive control), LPS (1 μg/mL; positive control) or CVX (5 and 10 μg/mL) for 12 and 24 h. IL-2 (A,B) and IL-10 (C,D) concentrations were quantitated by EIA assay in the supernatant collected after incubation with RPMI, ConA or LPS or CVX in spectrophotometer. IL-10 release was also quantified in PBMCs pre-treated with laminarin (100 µg/mL) for 30 min and after incubated with RPMI (negative control) or CVX (10 μg/mL) (E). The results were expressed as pg/mL of cytokines liberated and represent the mean ± S.E.M of 3 donors. ^*P < 0.05 compared to negative control, #P < 0.05 compared to untreated PBMCs incubated with CVX (Data were presented with ANOVA followed by Tukey post-test).

Figure 4

Nitrite and ROS production in PBMCs in the presence of Convulxin. NO quantification in the supernatant of 2 × 10⁵ PBMCs and ROS determination in 1 × 10⁶ CD³⁺ and CD⁴⁺ cells with DCFDA were quantified by flow cytometer followed by stimulation with RPMI without phenol red (negative control), PMA (500 ng/mL; positive control) or CVX (5 and 10 μg/mL) for selected time intervals. NO concentrations were quantitated by Griess method in supernatant collected after incubation with RPMI, LPS or CVX. ROS were determined in CD³⁺ and CD¹⁴⁺ gated populations following the addition of DCFDA (10 μM), and the DCF fluorescence was determined in FL1 channel in FACScan. The results were expressed as μM of NO liberated (A–C) and % of cell producing ROS and represent the mean ± S.E.M of 3 donors (D–K). ^*P < 0.05 compared to negative control (Data were presented with ANOVA followed by Tukey post-test). The images were collected using constant automatic gain among the samples to quantify the differences in absolute levels of fluorescence intensity of different conditions in 100 × magnification oil immersion objective. Figure representative of one experiment of three independent experiments (L,M). Analysis of the mean fluorescence intensity of ROS immunofluorescence was performed using 10 cells in field of view of each condition collected impartially.

Figure 5

Protein expression of inflammasome NLRP3 in PBMCs in the presence of Convulxin. Western blot of NLRP3, Pro-Caspase-1, Active Caspase-1, IL-1β, and β-actin (control) using 1 × 10⁷ PBMCs stimulated RPMI (negative control), LPS (1 μg/mL; positive control) or CVX (5 and 10 μg/mL) for 3 h. (A) Figure representative of one experiment of three independent experiments. Relative immunoreactivity analysis (fold of β-actin) of the western blots from NLRP3, Pro-Caspase-1, Active Caspase-1, IL-1β (B). IL-1β release was also quantified in PBMCs pretreated with N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (30 µM), Ac-YVAD-cmk (50 µM) (C), MCC950 (10 µM) (D) for 30 min and after incubated with RPMI (negative control) or CVX (10 µg/mL). IL-1β was also quantified in PBMCs pretreated with Apocynin (300 µM) and Rotenone (10 µM) for 30 min and after incubated with RPMI (negative control) or CVX (10 µg/mL) (E). Results were expressed in pg/mL of released cytokines and represent the mean ± S.E.M of 3 donors. Values are mean S.E.M. from 3 donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to negative control (data were presented with ANOVA followed by Dunnett post-test).

Figure 6

Complexes formed in the interaction of CVX (β subunits in orange and α subunits in sandy brown) with ectodomains of C-type Lectins receptors CLEC4E (red) and Dectin-2 (yellow). The complexes displayed above are the extracted central structures from the largest cluster of each respective MD trajectory, both structures are approximations of the most predominant conformations assumed by these proteins during 10 ns of simulation. The ΔG in kcal/mol of each interaction interface is highlighted in lines colored according to its complex of origin. Residues coordinating the interaction interfaces are highlighted on the interaction maps with hydrogen bonds depicted in green dashed lines and hydrophobic interactions highlighted in red protrusions.

Figure 7

Molecular dynamics strategies of CVX/CLEC4E and CVX/Dectin-2 simulations. The graphs above exhibit the 10 most populated clusters (grey bars) extracted from the MD trajectories of CVX/CLEC4E and CVX/Dectin-2 simulations, relating the number of members per cluster and the variation in binding energy among the representants (central structure) of each cluster (red and yellow lines).

Figure 8

Backbone RMSD measures from the trajectories of CVX/CLEC4E (red) and CVX/Dectin-2 (yellow) simulations. To avoid any masking effect generated by the hole CVX (αβ)₄ tetramer the RMSD was measured only from the main interacting parties, as highlighted in the image above.

Figure 9

Superposition of Rhodocytin in complex with CLEC-2 (gray) crystal structure (PDB: 3WWK) and the complexes predicted for the interactions between CVX (β subunits in orange and α subunits in sandy brown) and the CTLRs CLEC4E (red) and Dectin-2 (yellow).

Figure 10

The mechanism of the CVX interaction with the CTLR proposed (Dectin) in the present study. The interaction of CVX with CTLR (1) induces ROS production (2) and NF-κB activation (3). ROS induce the activation of the NLRP3 inflammasome complex (4); stimulating the production (5) and release of the pro-inflammatory cytokine IL-1β (6). The IL-1β production by the interaction of CVX and CTLR, induces the transcription of IL-10 (7); and consequently, its release (8). The interaction of IL-10 with its specific receptor (9), induces JAK-1 phosphorylation (10), initiating signaling via STAT3 (11,12), performing an anti-inflammatory activity, inhibiting transcription (13), production (14) and IL-2 release (15).