*3.2. Immunomodulation Properties*

Immunological behavior of spleen-derived immunocompetent cells and RAW 264.7 cells exposed to PIPOx has been followed up according to inductive release of signature Th1 (IFN-γ), Th2 (IL-4), Th17 (IL-17) and Treg (IL-10) cytokines and effectivity of phagocytic activity.

The isolated spleen cells were divided into the cell populations using the sequential adherence technique and 4 different groups enriched with distinct types of immunocytes were obtained as described in Table 1. Subsequently, these cell populations were used for stimulation and co-stimulation experiments (Figures 3–5).

Cytokine profile analysis of culture media after splenocytes stimulated with PIPOx reveal statistically significant increase of IL-10 production, even higher compared to Con A stimulation. Increase of IFN-γ, IL-17 and IL-4 production was not induced by PIPOx stimulation of complete splenocytes (Figure 3). PIPOx stimulation of adherent spleen cells obtained after the 1st adherence period, enriched in CD11c<sup>+</sup> and CD14<sup>+</sup> antigenpresenting cells (APCs), induced significantly enhanced production of IFN-γ (72 times higher compared to unstimulated control and 4.4 times higher than Con A) and increase in production of IL-17 (2.4 times higher compared to unstimulated control but 20 times lower than Con A). The cell-release of IL-4 and IL-10 was not affected. Obtained results indicate direction of the immune response polarization towards Th1/Th17 over Th2 and Treg immune responses (Figure 3). Adherent spleen cells following the 2nd adherence period, more enriched in CD11c<sup>+</sup> APCs, upon stimulation with PIPOx produced statistically

significantly higher amount of IL-10 (1.8 times higher in comparison with unstimulated control although 1.3 times lower than Con A) indicating Treg polarization of immune response (Figure 3).

**Figure 3.** Production of cytokines after in vitro stimulation. Concentrations of IFN-γ, IL-17, IL-4 and IL-10 in media after stimulation of splenocytes and adherent spleen cells after the 1st (SPL/a1) and the 2nd (SPL/a2) adherence period. Response to stimulation with Con A (10 μg/mL) and PIPOx (5 mg/mL). As a negative control, media of unstimulated cells were used. All data are shown as mean ± SD and statistical significance of differences between Con A or PIPOx-stimulated cells and unstimulated cells are expressed: \*\*\*—*p* < 0.001, \*\*—0.001 < *p* < 0.01, \*—0.01 < *p* < 0.05. Tests were performed in triplicate.

Adherent spleen cells, enriched in APCs, were pulsed with PIPOx or Con A and cocultured with non-adherent splenocytes (increased proportion of T-lymphocytes). Culture media after co-cultivation were used for cytokines analysis (Figure 4). PIPOx induced statistically significant increase of IFN-γ production (14.9 times higher than unstimulated APCs, but 8 times lower compared to Con A), IL-17 production (5.7 times higher in comparison with unstimulated APCs although 42.8 times lower compared to Con A), IL-10 production (5.0 times higher than unstimulated APCs and 2.1 times lower compared to Con A) and slight statistically non-significant increase of IL-4 production (1.8 times higher than unstimulated APCs and 79.1 times lower compared to Con A) in cultures containing pulsed adherent spleen cells population following the 1st adherence period (Figure 4). Adherent spleen cells after the 2nd adherence period pulsed with PIPOx did not induced significant increase of cytokines production after co-cultivation with splenic non-adherent cells (Figure 4).

Splenocytes stimulated with PIPOx were analyzed for production of Th1, Th2, Th17 and Treg signature cytokines via flow cytometry (Figure 5). PIPOx exposition induced increase of CD4<sup>+</sup> producing cells for all monitored cytokines. Results reveal higher percentage of IFN-γ (1.43 times higher than control) and IL-10 (1.92 times higher than control) producing CD4+ T-lymphocytes compared to CD4+IL-4+ (1.85 times higher than control) and CD4+IL-17+ (2.33 times higher than control) cells (Figure 5). Induction of cytokine producing cells upon stimulation with PIPOx was for IFN-γ, IL-10, and IL-4 lesser than Con A stimulation, except 1.74 times higher proportion of IL-17 producing CD4+ cells induced by PIPOx in comparison with Con A stimulation.

**Figure 4.** Production of cytokines after in vitro co-stimulation. Concentration of IFN-γ, IL-17, IL-4 and IL-10 in media after co-stimulation of non-adherent spleen cells with stimulated adherent spleen cells obtained after the 1st (SPL/a1) and the 2nd (SPL/a2) adherence period. Response to stimulation with Con A (10 μg/mL) and PIPOx (5 mg/mL). As a negative control, media of unstimulated cells were used. Tests were carried out in triplicate. The experimental data are expressed as geometric means of three replicates ± SD. Levels of significance: \*\* 0.001 < *p* < 0.01, \* 0.01 < *p* < 0.05. Differences were considered significant at 0.01 < *p* < 0.05.

**Figure 5.** Flow-cytometric analysis of cytokine producing CD4+ lymphocytes. Distribution of IFN-γ, IL-4, IL-10 and IL-17 producing CD4<sup>+</sup> cells within spleen cells stimulated with Con A (10 μg/mL) and PIPOx (5 mg/mL). Tests were done in triplicate. The experimental data are expressed as geometric means of three replicates ± SD. Levels of significance: \*\* 0.001 < *p* < 0.01, \* 0.01 < *p* < 0.05. Differences were considered significant at 0.01 < *p* < 0.05.

PIPOx stimulation of adherent CD11c<sup>+</sup> and CD14+ spleen cells induced significantly enhanced production of IFN-γ and increased production of IL-17 thus indicating polarization of immune response towards Th1 (IFN-γ, *p* < 0.001) and Th17 (IL-17, *p* < 0.01) over Th2(IL-4, ns) and Treg(IL-10, ns) immune responses. Adherent spleen cells more enriched in CD11c+ APCs produced significantly higher amount of IL-10 indicating Treg polarization of immune response.

Flow-cytometric analysis of cytokine producing CD4+ lymphocytes reveal higher proportion of IFN-γ and IL-10 producing CD4+ T-lymphocytes compared to CD4+IL-4+ and CD4+IL-17+ cells. Evidently, according to the PIPOx induced production of Th1, Th2, Th17and Treg signature cytokines, pro-inflammatory Th1 (IFN-γ, *p* < 0.001) and

Th17 (IL-17, *p* < 0.01) immune responses are more profound in exposed adherent CD11c<sup>+</sup> and CD14<sup>+</sup> spleen cells, the adherent spleen cells with higher expression CD11c<sup>+</sup> exert anti-inflammatory Treg (IL-10, *p* < 0.05) polarized response.

The immunocytometric analysis of RAW 264.7 macrophages exposed to different concentration of PIPOx revealed no significant impact on effective phagocytosis of *C. albicans*–FITC complex (Figure 6). Thus, the phagocytic capacity and functional effectivity of RAW 264.7 macrophages remained upon treatment with PIPOx without any significant impacts.

**Figure 6.** RAW 264.7 macrophage phagocytosis of *C. albicans*–FITC (%) following treatment with PIPOx analyzed by flow cytometry. The phagocytosis of FITC-labeled *C. albicans* cells by RAW 264.7 macrophages was analyzed by pre-treatment of cells for 1, 3, 6, 20, and 24 h with 0.5 mg/mL (**A**), and 5 mg/mL (**B**) of PIPOx and subsequent phagocytosis test analyzed using flow cytometry. Control represents untreated cells (Control). All data are expressed as Mean ± SD. Tests were carried out in triplicate. The one-way ANOVA and post hoc Bonferroni's tests was used to determine statistical significance of differences between untreated and stimulated cells and is expressed as ns—*p* > 0.05.

#### *3.3. Cell Internalization and Organelles Tracking Study*

The time and concentration-dependent PIPOx cell-processing (Figure 7) revealed sequential highly concentration-dependent intracellular accumulation of PIPOx. The highest intracellular accumulation of PIPOx for concentration 0.05 mg/mL was observed after 3 h exposition (68.9 times higher compared to the control), for concentration 0.1 mg/mL after 6 h treatment (120.9 times higher compared to the control) and for concentration 0.5 mg/mL after 3 h treatment (271.2 times higher compared to the control). The maximal PIPOx internalization, observed for the highest concentration (0.5 mg/mL), markedly exceeded (3.94-fold) the internalization of the lowest PIPOx concentration (0.05 mg/mL).

Using the flow cytometry, we have clearly demonstrated the uptake of PIPOx into the macrophages. Furthermore, the localization of PIPOx within the macrophage cell was performed using the organelle tracking dyes and fluorescently labeled PIPOx-FITC. The PIPOx-FITC treatment and staining with organelle tracking dyes was followed by ROI analysis according to Pearson and colocalization was expressed by correlation coefficient (R). Colocalization of PIPOx-FITC with organelle is considered when R > 0.5. PIPOx-FITC is localized within the macrophage in vesicular particles as seen on CLSM images (Figure 8). Based on ROI analysis, these vesicles containing PIPOx-FITC do not co-localize with mitochondrial network as R = 0.3 ± 0.1 (Figure 8A,C). Partial colocalization of PIPOx-FITC containing vesicles with lysosomes stained with Lyso Tracker (Figure 8B,D) with R = 0.5 ± 0.16 is observed. There are many vesicles containing PIPOx that do not co-localize with lysosomes.

**Figure 7.** Flow-cytometric analysis of time kinetics and concentration-dependent PIPOx-FITC processing by RAW 264.7 macrophages. The PIPOx-FITC cell bound and internalized by RAW 264.7 macrophages was analyzed by exposition of cells for 1, 3, 6, 20, and 24 h with 0.05 mg/mL, 0.1 mg/mL and 0.5 mg/mL or FITC-labeled PIPOx using flow cytometry. The internalized PIPOx-FITC was analyzed after Trypan blue quenching. The amount of membrane attached PIPOx-FITC is expressed as a difference between the cells with cell bound and internalized PIPOx-FITC and Trypan blue quenched cell population (internalized PIPOx-FITC). Cell bound and internalized, membrane attached and internalized PIPOx-FITC were expressed as mean fluorescence intensities (MFI) of 10,000 analyzed cells. All data are shown as Mean ± SD. Tests were done in triplicate. The statistical significance of differences between control cells and stimulated cells by means of one-way ANOVA and post hoc Bonferroni's test is presented as: \*\*\*—*p* < 0.001, \*\*—0.001 < *p* < 0.01, \*—0.01 < *p* < 0.05.

**Figure 8.** CLSM imaging and ROI analysis of PIPOx-FITC colocalization with organelles. ROI analysis in (**A**) mitochondria, R = 0.3 ± 0.1, and (**B**) lysosomes, R = 0.5 ± 0.16. Merged CLSM images of fluorescently labeled PIPOx-FITC (green) with (**C**) MitoTracker-labeled mitochondria (orange) or (**D**) LysoTracker-labeled lysosomes (red).
