*2.4. BaV Induces the Production of TNF and IL-1β in THP-1 Macrophages*

The concentration of cytokines IL-1β, IL-6, IL-10, IL-12, and TNF were evaluated on cell-free supernatants by CBA (Cytometric Bead Array) after 24 h, 48 h, and 72 h of treatments with BaV. As shown in Figure 1, BaV induced the production of TNF and IL-1β in a concentration-dependent manner. The production of TNF was detected only after 24 h in response to the two highest doses of BaV (10 μg and 15 μg/well). On the other hand, the maximum production of IL-1β, also in response to the two highest doses of BaV, was reached in 24 h, then decreased after 48 h and 72 h of treatment. BaV did not induce the production of IL-6, IL-10, or IL-12.

**Figure 1.** Cytokine production in THP-1 macrophages induced by BaV. THP-1 macrophages (2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) were treated with different concentrations of BaV (0.5 to 15 μg/well) to induce cytokine production. Cells incubated only with culture medium were used as control. After 24 h, 48 h, and 72 h, the cytokine production was evaluated in culture supernatant by CBA. (**a**) TNF and (**b**) IL-1β. Results expressed as mean of duplicates ± SEM and analyzed by one-way ANOVA followed by Tukey's post-test (each period compared with respective control) or two-way ANOVA followed by Tukey's post-test (comparison between different periods of treatment). (\*) Significant difference in relation to the respective control. (#) Significant difference between concentrations of BaV on each period. Symbols indicate differences between periods of treatment: (α) difference of 48 h or 72 h compared to 24 h; (β) difference from 72 h to 48 h. *p* < 0.01 (\*\*); *p* < 0.001 (\*\*\*); and *p* < 0.0001 (\*\*\*\* and ####).

#### *2.5. BaV Induces the Production of RANTES and IL-8 in THP-1 Macrophages*

As the two highest doses of BaV (10 and 15 μg/well) were able to induce the cytokines production without affecting the cell viability, these doses were selected to evaluate the production of chemokines CXCL8/IL-8, CCL5/RANTES, CXCL9/MIG, CCL2/MCP-1, and CXCL10/IP-10 in cell-free supernatants by CBA after 24 h, 48 h, and 72 h of treatments with BaV. BaV induced the production of RANTES and IL-8. RANTES was produced in a dose-dependent manner after 24 h, decaying thereafter. IL-8, in contrast, started to be produced—also in a dose-dependent manner—mainly after 48 h and continuing after 72 h (Figure 2). BaV did not induce the production of CXCL9/MIG, CCL2/MCP-1, and CXCL10/IP-10.

#### *2.6. Kn-Ba Induces the Production of TNF, IL-6, and IL-1β in THP-1 Macrophages*

The concentrations of cytokines IL-1β, IL-6, IL-10, IL-12, and TNF were evaluated on cell-free supernatants by CBA after 24 h, 48 h, and 72 h of treatments with Kn-Ba. Kn-Ba induced the production of TNF and IL-1β, and, unlike BaV, it was also able to induce IL-6 production. At the two doses used (0.5 and 1 μg), Kn-Ba induced the production of these chemokines in concentration-dependent manners. The TNF and IL-6 production were detected after 24 h, decreasing thereafter, whereas the production of IL-1 β was accentuated after 48 h and maintained after 72 h (Figure 3). Kn-Ba did not induce the production of IL-10 or IL-12.

### *2.7. Kn-Ba Induces the Production of IP-10, MCP-1, RANTES, and IL-8 in THP-1 Macrophages*

The concentrations of chemokines CXCL8/IL-8, CCL5/RANTES, CXCL9/MIG, CCL2/ MCP-1, and CXCL10/IP-10 were evaluated in cell-free supernatants by CBA after 24 h, 48 h, and 72 h of treatments with Kn-Ba (0.5 and 1 μg). Just like BaV, Kn-Ba induced the production of RANTES and IL-8, but was also able to induce the production of IP-10 and MCP-1, in time- and concentration-dependent manners (Figure 4).

**Figure 2.** Chemokine productions in THP-1 macrophages induced by BaV. THP-1 macrophages (2 <sup>×</sup> 105 cells/well) were treated with two concentrations of BaV (10 and 15 <sup>μ</sup>g/well). Cells incubated only with culture medium were used as control. After 24 h, 48 h, and 72 h, the production of chemokines were evaluated in culture supernatant by CBA. (**a**) RANTES and (**b**) IL-8. Results expressed as mean of duplicates ± SEM and analyzed by one-way ANOVA followed by Tukey's post-test (each period compared with respective control) or two-way ANOVA followed by Tukey's post-test (comparison between different periods of treatment). (\*) Significant difference in relation to the respective control. (#) Significant difference between concentrations of BaV on each period. Symbols indicate differences between periods of treatment: (α) difference of 48 h or 72 h compared to 24 h; (β) difference from 72 h to 48 h. *p* < 0.05 (\*); *p* < 0.01 (##); *p* < 0.001 (\*\*\* and ###); and *p* < 0.0001 (\*\*\*\* and ####).

**Figure 3.** *Cont*.

**Figure 3.** Cytokine production in THP-1 macrophages induced by Kn-Ba. THP-1 macrophages (2 <sup>×</sup> 105 cells/well) were treated with two concentrations of Kn-Ba (0.5 and 1 <sup>μ</sup>g/well). Cells incubated only with culture medium were used as control. After 24 h, 48 h, and 72 h the production of cytokines were evaluated in culture supernatant by CBA. (**a**) TNF, (**b**) IL-6 and (**c**) IL-1β. Results expressed as mean of duplicates ± SEM and analyzed by one-way ANOVA followed by Tukey's post-test (each period compared with respective control) or two-way ANOVA followed by Tukey's post-test (comparison between different periods of treatment). (\*) Significant difference in relation to the respective control. (#) Significant difference between concentrations of Kn-Ba on each period. Symbols indicate differences between periods of treatment: (α) difference of 48 h or 72 h compared to 24 h; (β) difference from 72 h to 48 h. *p* < 0.01 (\*\* and ##); *p* < 0.001 (###); and *p* < 0.0001 (\*\*\*\* and ####).

**Figure 4.** Chemokine productions in THP-1 macrophages induced by Kn-Ba. THP-1 macrophages (2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) were treated with two concentrations of Kn-Ba (0.5 and 1 μg/well). Cells incubated only with culture medium were used as control. After 24 h, 48 h, and 72 h, the production of chemokines were evaluated in culture supernatant by CBA. (**a**) IP-10, (**b**) MCP-1, (**c**) RANTES and (**d**) IL-8. Results expressed as mean of duplicates ± SEM and analyzed by one-way ANOVA followed by Tukey's post-test (each period compared with respective control) or two-way ANOVA followed by Tukey's post-test (comparison between different periods of treatment). (\*) Significant difference in relation to the respective control. (#) Significant difference between concentrations of Kn-Ba on each period. Symbols indicate differences between periods of treatment: (α) difference of 48 h or 72 h compared to 24 h; (β) difference from 72 h to 48 h. *p* < 0.01 (\*\*); *p* < 0.001 (###); and *p* < 0.0001 (\*\*\*\* and ####).
