*2.3. In Vitro Pro-Inflammatory Effects of TsHpt I and -II on Mouse Macrophages*

After 24 h of incubation, TsHpt-I and -II exerted intermediate cytotoxicity on naïve murine peritoneal macrophages in vitro, as demonstrated by the statistically significant decrease in cell viability, to about 75% and 60%–70% at concentrations of 10 μg/mL and 50 μg/mL, respectively, when compared to 100% in the control group (medium), as determined by the MTT method (Figure 3). Cytotoxicity was not shown to be dosedependent (Figure 3).

Despite the moderate cytotoxic effect in the concentrations of 10 μg/mL and 50 μg/mL, TsHpt-I and -II were not able to decrease cellular viability in the higher concentration of 100 μg/mL. Interestingly, in this same concentration, both peptides were able to promote the release of significant levels of TNF, and TsHpt-II also induced the release of IL-6 (Figure 4). None of the peptides were able to induce the release of IL-10, IL-12p70, IFN-γ and MCP-1 (data not shown).

In order to confirm the pro-inflammatory effects of both peptides on the biological function of macrophages, the phagocytic activity of macrophages, cultured for 24 h in the presence of TsHpt-I and -II, were analyzed, and compared to the negative and positive controls. Both treatments with TsHpt-I and -II promoted a significant increase in the phagocytic index (Figure 5), demonstrating that the pro-inflammatory action of these peptides also affects the biological function of macrophages.

**Figure 3.** Cytotoxic effect of hypotensins I and II on murine peritoneal macrophages in vitro. Peritoneal exudate macrophages from naïve BALB/c mice were cultured for 24 h in the presence of TsHpt-I (**A**) and -II (**B**) at concentrations of 10 μg/mL, 50 μg/mL and 100 μg/mL. Cells cultured with medium alone or in the presence of 5 μg/mL LPS were used as the controls. Cell viability was determined by the MTT method. The results represent the mean ± SD of two independent experiments performed in triplicate. \* *p* < 0.05.

**Figure 4.** Cytokine production by murine peritoneal macrophages stimulated in vitro with TsHpt-I and -II. Peritoneal exudate macrophages from naïve BALB/c mice were cultured for 24 h in the presence of TsHpt-I (**A**,**C**) and -II (**B**,**D**), at concentrations of 10 μg/mL, 50 μg/mL and 100 μg/mL. Cells cultured with medium alone or in the presence of 5 μg/mL LPS were used as negative and positive controls, respectively. The concentrations of TNF (**A**,**B**) and IL-6 (**C**,**D**) were determined by the CBA method, according to the manufacturer's instructions. The results represent the mean ± SD of three independent experiments performed in triplicate. \* *p* < 0.05.

**Figure 5.** Phagocytic activity of the murine peritoneal macrophages stimulated in vitro with TsHpt-I and -II. Peritoneal exudate macrophages from naïve BALB/c mice were cultured on glass coverslips for 24 h in culture medium or in the presence of LPS (5 μg/mL) or TsHpt-I and -II (100 μg/mL). After this period, the cells were incubated for 1 h with a suspension of *Saccharomyces cerevisiae*. Coverslips were washed, fixed and stained by Giemsa. (**A**) Phagocytosis images in 400× magnification. (**B**) The phagocytic index was calculated based on the percentage of phagocytic cells and on the average number of yeasts per cell, by counting approximately 200 cells per coverslip in 1000× magnification. The results represent one of two reproducible experiments performed in duplicate (mean ± SD). \* *p* < 0.05.
