Immunomodulatory effect of Tityus sp. in mononuclear cells extracted from the blood of rheumatoid arthritis patients

Abstract

Background: Pathophysiological mechanisms of rheumatoid arthritis arise because of a proinflammatory environment, generated by the interaction of autoreactive lymphocytes and proinflammatory mediators. Current strategies to mitigate the progression of the disease produce adverse effects, so there is a need for new therapeutic strategies and molecular targets to treat this disease. In this context, evidence suggests that scorpion venoms could modulate the immune response and some important cellular mechanisms of pharmacological interest. To evaluate the immunomodulatory effect of the venom of Tityus sp. (a possible new species close to Tityus metuendus) peripheral blood mononuclear cells of women diagnosed with RA were compared to cells of a control group.

Methods: A case-control study was conducted with a sample of 10 women with a confirmed diagnosis of RA and controls matched by sex and age. The cytotoxicity of the venom was evaluated to find sublethal concentrations of the venom, and subsequently, their immunomodulatory capacity in terms of percentage of proliferation, cell activation, and cytokines production.

Results: the venom of Tityus sp. produced a decrease in the percentage of proliferation in the CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ cell subpopulations of RA patients and healthy controls, at concentrations of 252 and 126 µg/mL. However, the venom did not induce significant differences in the percentage of cell activation markers. The venom caused a decrease in IL-10 at a concentration of 252 µg/mL compared to untreated cells from patients and controls. The remaining cytokines did not show significant differences.

Conclusion: the venom of Tityus sp. is a potential source of molecules with immunomodulatory ability in CD4 and CD8 T lymphocytes. This result directs venom characterization studies to identify pharmacological targets with immunomodulatory capacity in T lymphocytes to enhance research in the treatment of autoimmune disorders such as RA.

Keywords: Activation; Flow cytometry; Immunomodulatory activity; Proliferation; Rheumatoid arthritis; T lymphocytes; Tityus aff. metuendus; Tityus sp.; Venom.

Figure 1.. Effect of Tityus sp. venom (VTsp) on the cell viability of peripheral blood mononuclear cells (PBMC), that were stimulated with PHA (Phytohemagglutinin), and exposed to the VTsp in a 72h culture, using the resazurin fluorimetric assay (AlamarBlue). Negative control: Triton 1x; positive control: untreated cells. Results were expressed as the standard error of the mean (± SEM). Significance values p < 0.05* and p < 0.01**.

Figure 2.. Percentage of cell activation of peripheral blood mononuclear cells (PBMC) that were stimulated with PHA (Phytohemagglutinin), and exposed to the VTsp, in a 72h culture. Activation analysis of (A) CD4⁺CD69⁺, (B) CD4⁺HLADR⁺, (C) CD8CD69⁺, (D) CD8⁺HLADR⁺, cell populations exposed to Tityus sp. venom (252, 126, and 63 µg/mL) comparing RA patients and healthy controls.

Figure 3.. Effect of Tityus sp. venom on the percentage of PBMC proliferation; (A) Dotplot representative of the cytometric analysis to assess the percentage of cell proliferation in the CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ populations that were stimulated with PHA (Phytohemagglutinin), marked with CFSE, and exposed to Tityus sp. venom in a 72-hour culture; (B) Representative histogram of the effect of Tityus venom on PBMC proliferation. The arrows refer to the number of cell cycles that occurred during the 72 hours.

Figure 4.. Percentage of the proliferation of PBMC treated with Tityus sp. venom. Proliferation analysis of (A) CD3⁺, (B) CD3⁺CD4⁺, (C) CD3CD8⁺ CFSE^low cell populations, which were stimulated with PHA (Phytohemagglutinin) and exposed to Tityus sp. venom (252, 126 and 63 µg/mL) comparing RA patients and healthy controls. Results are expressed as mean standard error (± MSE). Significance value p < 0.01**.

Figure 5.. Analysis of cytokine expression of PBMC from RA patients and healthy controls exposed to VTsp venom. Production of cytokines (A) IL-6, (B) TNF-α, (C) IL1-β, and (D) IL-10 expressed in pg/mL of mononuclear cells from patients diagnosed with RA and healthy patients cultured with PHA and treated with Tityus sp. Results are expressed as the mean standard error (± MSE). Significance values were p < 0.01** and p < 0.05*.