*2.2. Effect of PF on Cytokine and Chemokine Production by CD4<sup>+</sup> T Cells*

To reveal whether the PF from the patients with endometriosis and the control subjects may affect the cytokine and chemokine production by CD4<sup>+</sup> T cells, we evaluated cytokine and chemokine production following the 5-day culture of unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells, where stimulation with CD3/CD28 beads mimics antigen stimulation conditions [27,28]. The results of IL-2, IFN-γ, IL-17A, TNF, IL-4, IL-10 and IL-6 concentrations in CD4<sup>+</sup> T cell culture supernatants are shown in Figure 1. As can be seen, CD4<sup>+</sup> T cell stimulation with CD3/CD28 beads and IL-2 resulted in the very high upregulation of production of all tested cytokines as compared to unstimulated cells. The addition of the endometriotic PF to the culture of CD4<sup>+</sup> T cells revealed its suppressive effect on the production of IL-2 (Figure 1A), IFN-γ (Figure 1B), IL-17A (Figure 1C) and TNF (Figure 1D), particularly by stimulated cells. Control PF also displayed some inhibitory activity toward the production of these cytokines; however, a statistically significant inhibition was seen only in the case of IL-2 production (Figure 1A). On the other hand, endometriotic PF significantly upregulated production of IL-4 (Figure 1E) and IL-10 (Figure 1F) by stimulated CD4<sup>+</sup> T cells. The production of IL-10 by stimulated CD4<sup>+</sup> T cells was also upregulated by the control PF (Figure 1F). The production of IL-6 by unstimulated CD4<sup>+</sup> T cells was significantly stimulated by endometriotic PF, whereas the production of this cytokine by stimulated lymphocytes was affected by neither control nor endometriotic

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PFs (Figure 1G). There were no significant differences between PF from endometriosis and control group. otic PFs (Figure 1G). There were no significant differences between PF from endometriosis and control group.

also upregulated by the control PF (Figure 1F). The production of IL-6 by unstimulated CD4<sup>+</sup> T cells was significantly stimulated by endometriotic PF, whereas the production of this cytokine by stimulated lymphocytes was affected by neither control nor endometri-

**Figure 1.** Production of (**A**) IL-2, (**B**) IFN-γ, (**C**) IL-17A, (**D**) TNF, (**E**) IL-4, (**F**) IL-10 and (**G**) IL-6 by cultured CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2 in the presence of the culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF). The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired nonparametric ANOVA (Friedman's test) followed by a post hoc test. \*Statistically significant from the control group at least at *p* < 0.05. Baseline concentration ranges of the tested cytokines (pg/mL), respectively, in Control PF and ENDO PF used for the experiments were as follows. IL-2, 0.13–0.56 and 0.13–0.88; IFN-γ, 0–0.82 and 0–0.95; IL-17A, 0–8.10 and 0–8.70; TNF, 0.43–1.32 and 0.38–2.24; IL-4, 0–0.70 and 0–0.59; IL-10, 0.59–9.65 and 1.46–11.2; IL-6, 16.4–312.8 and 47.1–492. **Figure 1.** Production of (**A**) IL-2, (**B**) IFN-γ, (**C**) IL-17A, (**D**) TNF, (**E**) IL-4, (**F**) IL-10 and (**G**) IL-6 by cultured CD4+ T cells unstimulated or stimulated with CD3/CD28 beads and IL-2 in the presence of the culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF). The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired non-parametric ANOVA (Friedman's test) followed by a post hoc test. \* Statistically significant from the control group at least at *p* < 0.05. Baseline concentration ranges of the tested cytokines (pg/mL), respectively, in Control PF and ENDO PF used for the experiments were as follows. IL-2, 0.13–0.56 and 0.13–0.88; IFN-γ, 0–0.82 and 0–0.95; IL-17A, 0–8.10 and 0–8.70; TNF, 0.43–1.32 and 0.38–2.24; IL-4, 0–0.70 and 0–0.59; IL-10, 0.59–9.65 and 1.46–11.2; IL-6, 16.4–312.8 and 47.1–492.

The results of the evaluation of the effects of PF on chemokine (CCL2, CCL5, CXCL8 and CXCL9) production by CD4<sup>+</sup> T cells are shown in Figure 2. As seen, CD3/CD28/IL-2 stimulation of CD4<sup>+</sup> T cells significantly affected the production of all chemokines except CXCL8. It should be stressed, however, that production of the latter was extremely relatively high even in unstimulated CD4<sup>+</sup> T cells. The production of CCL2 was significantly upregulated in both unstimulated and stimulated CD4<sup>+</sup> T cells by the control as well as endometriotic PF (Figure 2A). The stimulation of CXCL8 production was seen only with endometriotic PF in unstimulated CD4<sup>+</sup> T cells (Figure 2C). On the other hand, both control and endometriotic PF significantly inhibited the production of CCL5 (Figure 2B) and CXCL9 (Figure 2D) by stimulated CD4<sup>+</sup> T cells. There were no significant differences be-The results of the evaluation of the effects of PF on chemokine (CCL2, CCL5, CXCL8 and CXCL9) production by CD4<sup>+</sup> T cells are shown in Figure 2. As seen, CD3/CD28/IL-2 stimulation of CD4<sup>+</sup> T cells significantly affected the production of all chemokines except CXCL8. It should be stressed, however, that production of the latter was extremely relatively high even in unstimulated CD4<sup>+</sup> T cells. The production of CCL2 was significantly upregulated in both unstimulated and stimulated CD4<sup>+</sup> T cells by the control as well as endometriotic PF (Figure 2A). The stimulation of CXCL8 production was seen only with endometriotic PF in unstimulated CD4<sup>+</sup> T cells (Figure 2C). On the other hand, both control and endometriotic PF significantly inhibited the production of CCL5 (Figure 2B) and CXCL9 (Figure 2D) by stimulated CD4<sup>+</sup> T cells. There were no significant differences between PF from endometriosis and control group.

tween PF from endometriosis and control group.

**Figure 2.** Production of (**A**) CCL2, (**B**) CCL5, (**C**) CXCL8 and (**D**) CXC9 by cultured CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2 in the presence of the culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF). The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired non-parametric ANOVA (Friedman's test) followed by a post hoc test. \* Statistically significant from the control group at least at *p* < 0.05. Baseline concentration ranges of the tested chemokines (pg/mL), respectively, in Control PF and ENDO PF used for the experiments were as follows. CCL2, 16.6–198.7 and 10.12–393.2; CCL5, 2.5–9.6 and 6.0–33.9; CXCL8, 7.08–61.98 and 15.4–400.0; CXC9, 12.1–50.8 and 39.2- 72.6. **Figure 2.** Production of (**A**) CCL2, (**B**) CCL5, (**C**) CXCL8 and (**D**) CXC9 by cultured CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2 in the presence of the culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF). The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired non-parametric ANOVA (Friedman's test) followed by a post hoc test. \* Statistically significant from the control group at least at *p* < 0.05. Baseline concentration ranges of the tested chemokines (pg/mL), respectively, in Control PF and ENDO PF used for the experiments were as follows. CCL2, 16.6–198.7 and 10.12–393.2; CCL5, 2.5–9.6 and 6.0–33.9; CXCL8, 7.08–61.98 and 15.4–400.0; CXC9, 12.1–50.8 and 39.2–72.6.

#### *2.3. Effect of PF on Generation of Treg and Th17 Cells 2.3. Effect of PF on Generation of Treg and Th17 Cells*

To see whether the PF from the patients with endometriosis and the control subjects may affect in vitro generation of Treg and Th17 cells we evaluated specific phenotype changes of the unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells following their 5-day culture. As seen in Figure 3, stimulation of CD4<sup>+</sup> T cells with CD3/CD28 beads and IL-2 resulted in significant generation of CD25high and CD25high FOXP3<sup>+</sup> Treg cells. The addition of the endometriotic PF significantly enhanced generation of the CD25high T cells as compared to culture medium control and PF from women without endometriosis. A similar effect of the endometriotic PF on the generation of CD25high FOXP3<sup>+</sup> cells was also seen in cultures of unstimulated CD4<sup>+</sup> T cells (Figure 3B), whereas there were no differences in the generation of CD25high FOXP3<sup>+</sup> cells in CD3/CD28/IL-2-stimulated cultures. Control PF did not affect generation of CD25high and CD25high FOXP3<sup>+</sup> T cells in either unstimulated or stimulated CD4<sup>+</sup> T cell cultures. To see whether the PF from the patients with endometriosis and the control subjects may affect in vitro generation of Treg and Th17 cells we evaluated specific phenotype changes of the unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells following their 5-day culture. As seen in Figure 3, stimulation of CD4<sup>+</sup> T cells with CD3/CD28 beads and IL-2 resulted in significant generation of CD25high and CD25high FOXP3<sup>+</sup> Treg cells. The addition of the endometriotic PF significantly enhanced generation of the CD25high T cells as compared to culture medium control and PF from women without endometriosis. A similar effect of the endometriotic PF on the generation of CD25high FOXP3<sup>+</sup> cells was also seen in cultures of unstimulated CD4<sup>+</sup> T cells (Figure 3B), whereas there were no differences in the generation of CD25high FOXP3<sup>+</sup> cells in CD3/CD28/IL-2-stimulated cultures. Control PF did not affect generation of CD25high and CD25high FOXP3<sup>+</sup> T cells in either unstimulated or stimulated CD4<sup>+</sup> T cell cultures.

Figure 4 shows that stimulation with CD3/CD28/IL-2 significantly increased the generation of CD161<sup>+</sup> T cells while having no significant effect on the generation of the CD161<sup>+</sup> RORγ + cells. Endometriotic PF had a significant suppressive effect on generation of CD161<sup>+</sup> , both in unstimulated and stimulated CD4<sup>+</sup> T cell populations (Figure 4A). Neither PF affected the generation of CD161<sup>+</sup> RORγ + cells (Figure 4B). There were no significant differences between PF from endometriosis and control group.

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**Figure 3.** Effect of culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF) on generation of CD25high and CD25high FOXP3<sup>+</sup> Treg cells in cultures of CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2. (**A**) Gating strategy and a representative flow cytometry analysis showing identification of the respective CD25high and CD25high FOXP3<sup>+</sup> T cell subpopulations in CD4<sup>+</sup> T cells under different culture conditions. (**B**) Proportions of CD25high T cells and (**C**) CD25high FOXP3<sup>+</sup> Treg cells in population of unstimulated or CD3/CD28 beads+IL-2-stimulated CD4<sup>+</sup> T cells. The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired (Friedman's test) or unpaired (Kruskal–Wallis test) non-parametric ANOVA followed by a post hoc test. Figure 4 shows that stimulation with CD3/CD28/IL-2 significantly increased the gen-**Figure 3.** Effect of culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF) on generation of CD25high and CD25high FOXP3<sup>+</sup> Treg cells in cultures of CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2. (**A**) Gating strategy and a representative flow cytometry analysis showing identification of the respective CD25high and CD25high FOXP3<sup>+</sup> T cell subpopulations in CD4<sup>+</sup> T cells under different culture conditions. (**B**) Proportions of CD25high T cells and (**C**) CD25high FOXP3<sup>+</sup> Treg cells in population of unstimulated or CD3/CD28 beads+IL-2-stimulated CD4<sup>+</sup> T cells. The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired (Friedman's test) or unpaired (Kruskal–Wallis test) non-parametric ANOVA followed by a post hoc test. in population of unstimulated or CD3/CD28 beads+IL-2-stimulated CD4<sup>+</sup> T cells. The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired (Friedman's test) or unpaired (Kruskal–Wallis test) non-parametric ANOVA followed by a post hoc test. Figure 4 shows that stimulation with CD3/CD28/IL-2 significantly increased the generation of CD161<sup>+</sup> T cells while having no significant effect on the generation of the CD161<sup>+</sup> RORγ<sup>+</sup> cells. Endometriotic PF had a significant suppressive effect on generation of CD161<sup>+</sup> , both in unstimulated and stimulated CD4<sup>+</sup> T cell populations (Figure 4A). Neither PF affected the generation of CD161<sup>+</sup> RORγ<sup>+</sup> cells (Figure 4B). There were no significant differences between PF from endometriosis and control group.

**Figure 4.** Effect of culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF) on generation of CD161<sup>+</sup> and CD161<sup>+</sup> RORγ <sup>+</sup> Th17 cells in cultures of CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2. (**A**) Gating strategy and a representative flow cytometry analysis showing identification of the respective CD161<sup>+</sup> and CD161<sup>+</sup> RORγ <sup>+</sup> T cell subpopulations in CD4<sup>+</sup> T cells under different culture conditions. (**B**) Proportions of CD161<sup>+</sup> T cells and (**C**) CD161<sup>+</sup> RORγ <sup>+</sup> Th17 cells in population of unstimulated or CD3/CD28 beads+IL-2-stimulated CD4<sup>+</sup> T cells. The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired (Friedman's test) or unpaired (Kruskal–Wallis test) non-parametric ANOVA followed by a post hoc test.

#### *2.4. Effect of PF on NK Cell Cytotoxicity 2.4. Effect of PF on NK Cell Cytotoxicity*

lowed by a post hoc test.

To reveal an effect of the PF from the patients with endometriosis and the control subjects on the NK cells, we evaluated the cytotoxic activity of the cultured PBMC against K562 erythroleukemia cells. As seen in Figure 5, a one day culture of PBMC with endometriotic PF resulted in a significant decrease in their cytotoxic activity. The control PF also displayed some inhibitory effect; this, however, was not statistically significant. There was also no significant difference between PF from endometriosis and control group. To reveal an effect of the PF from the patients with endometriosis and the control subjects on the NK cells, we evaluated the cytotoxic activity of the cultured PBMC against K562 erythroleukemia cells. As seen in Figure 5, a one day culture of PBMC with endometriotic PF resulted in a significant decrease in their cytotoxic activity. The control PF also displayed some inhibitory effect; this, however, was not statistically significant. There was also no significant difference between PF from endometriosis and control group.

**Figure 4.** Effect of culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF) on generation of CD161<sup>+</sup> and CD161<sup>+</sup> RORγ<sup>+</sup> Th17 cells in cultures of CD4<sup>+</sup> T cells unstimulated or stimulated with CD3/CD28 beads and IL-2. (**A**) Gating strategy and a representative flow cytometry analysis showing identification of the respective CD161<sup>+</sup> and CD161<sup>+</sup> RORγ<sup>+</sup> T cell subpopulations in CD4<sup>+</sup> T cells under different culture conditions. (**B**) Proportions of CD161<sup>+</sup> T cells and (**C**) CD161<sup>+</sup> RORγ<sup>+</sup> Th17 cells in population of unstimulated or CD3/CD28 beads+IL-2-stimulated CD4<sup>+</sup> T cells. The results are shown as scatter dot plots with a median and interquartile range. Statistical significance was computed by paired (Friedman's test) or unpaired (Kruskal–Wallis test) non-parametric ANOVA fol-

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**Figure 5.** Effect of culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF) on the NK cell cytotoxic activity of cultured PBMC. (**A**) The representative flow cytometry analysis showing the controls for the NK assay. Shown are the fluorescence-negative effector cells (PBMC), green fluorescence (FITC) labeled K562 target cells and spontaneously dying K562 cells (red fluorescence, PE). Spontaneous death of target cells was determined in cultures without effector cells. (**B**) An example of identification of target K562 cells killed by NK cells from the PBMC population (red fluorescence). (**C**) Relative cell mediated cytotoxicity of untreated and peritoneal fluid-treated PBMC against K562 cells. The results are expressed as an index of specific cytotoxicity of peritoneal fluid-treated PBMC relative to untreated control PBMC. Each bar represents mean ± SD from 4 independent experiments. Statistical significance was computed by paired non-parametric ANOVA (Friedman's test) followed by a post hoc test. **Figure 5.** Effect of culture medium alone (CM), peritoneal fluid from control woman (Control PF) or peritoneal fluid from woman with endometriosis (ENDO PF) on the NK cell cytotoxic activity of cultured PBMC. (**A**) The representative flow cytometry analysis showing the controls for the NK assay. Shown are the fluorescence-negative effector cells (PBMC), green fluorescence (FITC) labeled K562 target cells and spontaneously dying K562 cells (red fluorescence, PE). Spontaneous death of target cells was determined in cultures without effector cells. (**B**) An example of identification of target K562 cells killed by NK cells from the PBMC population (red fluorescence). (**C**) Relative cell mediated cytotoxicity of untreated and peritoneal fluid-treated PBMC against K562 cells. The results are expressed as an index of specific cytotoxicity of peritoneal fluid-treated PBMC relative to untreated control PBMC. Each bar represents mean ± SD from 4 independent experiments. Statistical significance was computed by paired non-parametric ANOVA (Friedman's test) followed by a post hoc test.

#### **3. Discussion 3. Discussion**

The results of the present study show for the first time that PF from women with advanced endometriosis displays immunomodulatory activity toward both unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells. We chose the stimulation of CD4<sup>+</sup> T cells with CD3/CD28 beads and IL-2 as this method is considered to be a good model for assessment of T cell receptor-dependent T cell activation and expansion [27,28]. We found The results of the present study show for the first time that PF from women with advanced endometriosis displays immunomodulatory activity toward both unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells. We chose the stimulation of CD4<sup>+</sup> T cells with CD3/CD28 beads and IL-2 as this method is considered to be a good model for assessment of T cell receptor-dependent T cell activation and expansion [27,28]. We found that endometriotic PF inhibited the production of IL-2, IFN-γ, IL-17A and TNF by CD4<sup>+</sup> T cells. On the other hand, it stimulated the production of IL-4 and IL-10. The production of IL-2, IFN-γ and TNF is a feature of the Th1 subpopulation of CD4<sup>+</sup> T cells, which is responsible for inflammatory and cell-mediated immunity, whereas the production of IL-4 and IL-10 is an attribute of Th2 cells, which are involved in the regulation of antibody production and the downregulation of cell-mediated responses [29,30]. Thus, our results may suggest that endometriotic PF displays an ability to shift CD4<sup>+</sup> T cell differentiation into the Th2 phenotype. This observation is in line with the previous suggestions that the Th1/Th2 balance is abrogated in the endometriosis patients and that Th2 cells may favor development of the disease [31,32]. It should be stressed, however, that our observations

were limited to cytokine evaluations and further studies on the shifting of the Th1/Th2 balance to Th2 phenotype are required.

We also found that the PF from women with endometriosis modulates the production of some T cell-derived chemokines. The treatment of CD4<sup>+</sup> T cells with endometriotic PF resulted in stimulation of CCL2 (also known as MCP-1) release. This is consistent with our observation that CCL2 concentrations are elevated in the PF from endometriosis patients as compared to healthy women (Table 2) as well as previous observations of many other investigators [33]. CCL2 is a key chemokine responsible for chemotaxis/infiltration and activation of monocytes/macrophages [34], thus, our present results strongly argue for the role of the PF milieu in generation of pelvic inflammation in the course of endometriosis. Interestingly, we also found that endometriotic PF inhibited the production of CCL5 (RANTES) and CXCL9 (MIG) by the CD4<sup>+</sup> T cells. CCL5 is responsible for the chemotaxis of T cells and some other leukocyte populations [35] and is considered to play a part in the pathogenesis of endometriosis [33]. CXCL9 is also responsible for T and NK cell infiltration, and, in particular, Th1 cells [36,37]. The expression of CXCL9 is upregulated by IFN-γ, thus, its downregulated production in endometriotic PF may reflect the inhibition of this cytokine release. These results seem to be in line with and extend the previous observations of Na et al. that endometriotic PF modulated production of CCL2, CCL3 (MIP-1α) and CCL5 by monocytes, neutrophils and T cells [38]. These findings strongly suggest that the PF from women with endometriosis displays immunosuppressive properties which may affect local infiltration and differentiation of T cells.

In addition to the observation that the PF from women with endometriosis affects cytokine/chemokine production by CD4<sup>+</sup> T cells. We also found that it stimulates differentiation/expansion of CD25high FOXP3<sup>+</sup> Treg cells. Increased numbers of Treg cells were repeatedly reported in the peritoneum of patients with endometriosis, thus suggesting their role in the suppression of the local immune responses [17,23,25,39,40]. Our present result suggests that the mechanism responsible for the increased numbers of Treg cells in the endometriotic PF may be at least partially due to their local activation by the peritoneal milieu.

A stimulatory effect of the endometriotic PF on generation of Treg cells was accompanied by an inhibition of expansion of CD161<sup>+</sup> Th17 cells. However, it should be noted that we did not observe any effect on the CD161<sup>+</sup> RORg<sup>+</sup> cells. It has been claimed that Th17 cells may play a part in the immunopathogenesis of endometriosis by exacerbation of the inflammatory response [41–43]. Nevertheless, our present observation suggests that the activity of Th17 cells may be suppressed by the factors present in the PF. Our observation might also explain the differential levels of Treg and Th17 cells in patients with different stage of the endometriosis [25].

Finally, we also confirmed the previous observations that the peritoneal fluid form women with endometriosis may also inhibit the cytotoxic activity of the NK cells [44,45].

Interestingly, the suppressive/modulatory effects of the PF of women with endometriosis were also reported on monocytes/macrophages. Accordingly, PF from the patients with endometriosis was reported to downregulate the expression of the MHC class II molecules as well as CD80 and CD86 costimulatory molecules in monocytes [46]. Furthermore, PF from women with endometriosis was also found to inhibit production of matrix metalloproteinases in the peritoneal macrophages [47].

Taken all together, our present results provide evidence that the PF of patients with endometriosis displays immunomodulatory/immunosuppressive activities toward CD4<sup>+</sup> T cells. These activities manifest by inhibition of Th1 and stimulation of Th2 cytokine production, the inhibition of some lymphocyte chemotactic factor production, the shift of the Treg/Th17 balance to Treg phenotype and the inhibition of NK cell cytotoxic activity. The nature of these modulatory/suppressive properties of the endometriotic PF remains a subject of speculations. It should be stressed, however, that unlike in comparison to culture medium control, there were no significant differences in immunomodulatory/immunosuppressive activities between endometriotic PF and PF from control

women without the disease. Furthermore, some inhibition of IL-2 and stimulation of IL-10 production by CD4<sup>+</sup> T cells was also seen in the case of PF from the control women. The control PF also stimulated CCL2 and inhibited CCL5 and CXCL9 production. This strongly suggests that PF from control women with ovarian dermoid cysts also displays some immunoregulatory activity. We included patients with ovarian dermoid cysts as control since this is a benign ovarian teratoma that typically does not manifest with local inflammatory response or systemic immune deviations [48]. Considering that the immunology of ovarian dermoid cysts remains elusive, the significance our present observation and the nature of this phenomenon remains to be elucidated.

The levels of some investigated cytokines and chemokines, such as IL-6, IL-10, CCL2, CXCL8, and CXCL9, were significantly increased in PF from patients with endometriosis as compared to control. This observation is consistent with a variety of previous reports [20,21,49,50] and argues for the role of endometriotic peritoneal milieu in the regulation of local inflammatory responses. It is tempting to speculate that the modulatory/suppressive activity of the endometriotic PF is at least partially attributable to the increased local production of some regulatory cytokines such as TGF-β and IL-10 [20]. Both cytokines exert strong anti-inflammatory activity and were found to be produced by and to facilitate the induction of Treg cells [51–54]. TGF-β may be also responsible for local inhibition of the NK cell activity [45]. It should be stressed, however, that the regulation of the Treg/Th17 balance cannot simply be explained by an excessive stimulation with TGF-β [55] and this issue requires further investigations. Similarly, it is also difficult to speculate about the possible mechanisms responsible for the change of the Th1/Th2 balance. The differentiation of both subpopulations of Th cells appears to be a complicated phenomenon depending on a bulk of immunoregulatory cytokines and accessory cells [56] and also deserves further study.

It should be stressed that due to difficulties in obtaining the sufficient amounts of PF for the research purposes, the present study was performed on a limited number of PF samples. Nevertheless, the present results suggest that the peritoneal milieu of women with endometriosis shows immunosuppressive properties and shifts the Th1/Th2 balance toward the Th2 phenotype. These properties may help us to understand how the endometrioid tissue may escape from under local immune surveillance. The shift of the Th1/Th2 balance may also account for the dysregulated control of antibody production and may explain the origin of endometriosis-associated autoimmune phenomena. This may strongly support the view that the peritoneal milieu plays an important part in the pathogenesis of endometriosis and may be a target for specific clinical interventions.
