*Article* **Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response**

**Joanna Olkowska-Truchanowicz <sup>1</sup> , Agata Białoszewska <sup>2</sup> , Aneta Zwierzchowska 3,4, Alicja Sztokfisz-Ignasiak <sup>2</sup> , Izabela Janiuk <sup>2</sup> , Filip D ˛abrowski 3,5 , Grazyna Korczak-Kowalska ˙ 6 , Ewa Barcz 3,4, Katarzyna Bocian 6,\* and Jacek Malejczyk 2,7,\***


**Abstract:** Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4<sup>+</sup> T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4<sup>+</sup> T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4<sup>+</sup> T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small

**Citation:** Olkowska-Truchanowicz, J.; Białoszewska, A.; Zwierzchowska, A.; Sztokfisz-Ignasiak, A.; Janiuk, I.; D ˛abrowski, F.; Korczak-Kowalska, G.; Barcz, E.; Bocian, K.; Malejczyk, J. Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response. *Int. J. Mol. Sci.* **2021**, *22*, 8134. https://doi.org/ 10.3390/ijms22158134

Academic Editors: Antonio Simone Laganà and Alfonso Baldi

Received: 23 May 2021 Accepted: 27 July 2021 Published: 29 July 2021

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number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.

**Keywords:** endometriosis; peritoneal fluid; cytokines; chemokines; Th1 cells; Th2 cells; Treg cells; Th17 cells
