*2.4. NO Production*

Stimulatory effects of algae polysaccharides on macrophages result in the production of NO through the induction of the enzyme inducible nitric oxide synthase [38,39]. NO is a highly reactive molecule that is important for the functioning of the immune system, and it has cytotoxic effects on pathogenic microorganisms and cancer cells [40]. Therefore, NO production in the supernatant of RAW 264.7 murine macrophages stimulated with *C. cupressoides* SPs was evaluated. The NO production induced by SP1 and SP2 at concentrations between 12.5 and 100 μg/mL, expressed in relation to the amount of NO produced in the positive control (2 μg/mL LPS), is shown in Figure 5. SP1 promoted a statistically significant increase (*p* < 0.05) in the production of NO at concentrations of 50 and 100 μg/mL, whereas for SP2, the concentrations of 25 and 50 μg/mL showed the greatest stimulatory effect on the production of NO. These results were similar to those reported in other works that evaluated the effects of SPs obtained from different species of seaweed on the ability of induce NO production in macrophages [41–43].

**Figure 5.** Effects of SP1 and SP2 of *C. cupressoides* on NO production. NC—negative control; LPS—bacterial lipopolysaccharide (2 μg/mL). The data demonstrate the mean ± standard deviation (*<sup>n</sup>*=3). Different letters represent statistically significant differences between SPs concentrations (*p*<0.05). Different numbers represent statistically significant differences between the same concentrations of different SPs (*p* < 0.05). \* represents samples that had a statistically significant difference in relation to the negative control (*p* < 0.05).

Studies on the immunostimulatory effect of polysaccharides have revealed that these molecules are capable of binding to several receptors on the surface of macrophages, which can activate several intracellular signaling pathways. As a result of this activation, the expression of genes that encode different inflammatory mediators, such as NO and cytokines, is initiated [44–46]. Some structural requirements, necessary for the immunostimulatory activity of algae polysaccharides, have been identified by Leiro et al. [47]. They found that the desulfation of polysaccharides from *Ulva rigida* was a determining factor for its immunostimulatory activity. The presence of the sulfate group was also shown to be an important characteristic for the effect of *Sargassum angustifolium* polysaccharides on NO production [48]. For SPs from *Ulva intestinales*, the lower molecular weight of one of the fractions was found to be a fundamental characteristic for immunostimulatory activity [13]. In another study, Bahramzadeh et al. [49] proposed that the compaction of the SPs obtained from *Cystoseira indica* seemed to be a more determinant characteristic for its immunostimulatory capacity than the size of the molecule and its degree of sulfation. However, the monosaccharide composition, sulfate content, and ultrastructure of *C. lentifera* polysaccharides appeared to have been crucial for their effect on NO production in RAW 264.7 macrophages [18]. Therefore, a systematic and in-depth study that aimed to relate the individual and/or combined structural characteristics of *C. cupressoides* SPs and their immunostimulatory effect was needed.

#### *2.5. Production of Intracellular ROS*

During the processes of the recognition and response to harmful cell agents, phagocytic cells produce NO and ROS [50]. Thus, ROS production is an important indicator of macrophage function [51]. Because the treatment of RAW 264.7 macrophages with the purified SPs of *C. cupressoides* promoted a stimulatory effect on NO production, it was determined whether there would be a similar effect on the production of intracellular ROS. Figure 6 shows the effect of treatment with SP1 and SP2 on the production of ROS after a 24 h exposure period at concentrations ranging from 12.5 to 100 μg/mL. The displacements of the histograms shown in A and B indicated that the two SPs promoted an increase in ROS production. Analysis of the fluorescence intensity emitted by cells treated with the SPs in relation to that of the positive control (2 μg/mL LPS) revealed a statistically significant

increase (*p* < 0.01) in all evaluated conditions, as seen in Figure 6C. These results are consistent with the effects of immunostimulating polysaccharides on ROS production reported in other studies. Wang et al. [52] reported that SPs from *Ascophyllum nodosum* significantly increased intracellular ROS levels in macrophages. In another study, sulfated galactans from the algae *Gracilaria lemaneiformis* also promoted stimulatory effects on ROS production [53], whereas the treatment of peritoneal macrophages with arabinogalactan and fucoidan increased the production of reactive oxygen and nitrogen species [54]. Although the increase in ROS production, induced by SP1 and SP2, was lower than that observed in the positive control, their stimulatory effects can be considered to be relevant and safe for macrophage activation because the excess production of ROS can lead to severe oxidative damage in cells.

**Figure 6.** Production of ROS. Histograms representative of the effects of different concentrations of SP1 (**A**) and SP2 (**B**) on intracellular ROS production quantified by flow cytometry. NC—negative control; LPS—bacterial lipopolysaccharide (2 μg/mL). (**C**) Percentage of intracellular ROS production in relation to that of LPS-stimulated cells. The data are presented as the mean ± standard deviation (*n* = 3). Different letters represent statistically significant differences between SPs concentrations (*p* < 0.01). Different numbers represent statistically significant differences between the same concentrations of different SPs (*p* < 0.01). \* represents samples that had a statistically significant differences in relation to the negative control (*p* < 0.01).

#### *2.6. Proinflammatory Cytokine Production*

Secretion of proinflammatory cytokines by activated macrophages is directly involved in the defense against pathogen invasion. These molecules play an important regulatory role in cell growth, proliferation, and immunity [55]. When exposed to immunostimulatory agents, macrophages secrete inflammatory mediators, including NO, ROS, and cytokines such as TNF-α and IL-6 [56,57]. Here, both cytokines were quantified in the supernatant of RAW 264.7 macrophages exposed to SP1 and SP2 at concentrations of 100 and 50 μg/mL, respectively. As shown in Figure 7A, a statistically significant increase (*p* < 0.01) of TNF-α production was observed in cells treated with SP1 and SP2, compared to that in the negative control. It should be noted that the effect of these SPs on the production of TNF-α was higher than that in the cells stimulated with LPS (positive control). SP1 and SP2 also promoted a statistically significant increase (*p* < 0.01) in the levels of IL-6, compared to that in the negative control, as seen in Figure 7B.

**Figure 7.** Production of the proinflammatory cytokines TNF-α (**A**) and IL-6 (**B**). NC—negative control; LPS—bacterial lipopolysaccharide (2 μg/mL). The data are presented as the mean ± standard deviation. \* represents samples that had a statistically significant differences in relation to the negative control (*p* < 0.01). # represents statistically significant increases (*p* < 0.01) in relation to LPS. SP1 and SP2 were evaluated at concentrations of 100 μg/mL and 50 μg/mL, respectively. IL-6 was not detected in the NC group.

The quantification of the proinflammatory cytokines TNF-α and IL-6 in macrophage cultures is an important assessment in studies on the immunomodulatory effects of polysaccharides obtained from algae. SPs from *Cyclocarya paliurus* increased the immunostimulatory activity of RAW macrophages through the production and secretion of various inflammatory mediators, including TNF-α and IL-6 [58]. In another study, the fucoidan from *Undaria pinnatifida* had a potent stimulatory effect on the production of TNF-α and IL-6 [59]. Similar effects on TNF-α and IL-6 secretion in RAW 264.7 macrophages were also observed in the study by Liu et al. [43] that evaluated the effects of *Porphyra haitanensis* SPs as well as in the work by Ren et al. [53] that evaluated the immunostimulatory effect of the polysaccharides from *Gracilaria lemaneiformis*. In a previous study, we showed that CCB-F1.0 promoted a significant increase in the production of IL-6 and TNF-α [24]. However, to investigate this effect, CCB-F1.0 was tested in concentrations 8 and 16 times higher than SP1 and SP2, respectively. These findings indicate that the purification process enabled isolation of the compounds with more proinflammatory effect. Therefore, *C. cupressoides* SPs may play an important role in stimulating the immune response mediated by macrophages.

#### **3. Materials and Methods**
