**7. Endometriosis and Inflammation**

Menstruation is an inflammatory process characterized by an increase in a variety of tissue-resident immune cells. A complex interaction between resident immune cells and uterine stromal cells modulates the biosynthesis and release of pro-inflammatory cytokines, chemokines, and prostaglandins (PGs), resulting in local vasoconstriction [3,6]. When retrograde menstruation occurs, endometrial fragments adhere and form lesions within the peritoneum. During this process, inflammatory cells are recruited to the lesions. This immune response is evident at lesion sites, with increased inflammatory cytokines/chemokines, growth factors, neutrophils and PGs found within the peritoneal cavity of EM patients [3,6,20,80,81].

Since EM is considered a chronic inflammatory disorder, the neuromodulatory mechanisms of the EM-associated immune cell infiltrate (EMaICI) need to be considered. In all investigated types of EM, immune cell infiltrates were observed and characterized as a mixture of several immunocompetent cells (T cells, B cells, and Mϕ) [31,82,83]. In endometriotic lesions and also in eutopic endometrial tissue of EM patients, the numbers of EMaICI were significantly higher than in control tissue and seem to be associated with a chronic inflammatory process [84]. EMaICI were observed and characterized in all the types of EM by our group as T lymphocytes (CD3+), helper T lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), antigen-experienced T lymphocytes "memory cells" (CD45RO+), macrophages (CD68+), and B lymphocytes (CD20+) [85]. The characterization of various types of immunocompetent cells in EMaICI demonstrated several distinct immunological reactions within the microenvironment of different endometriotic lesions.

Dendritic cells (DC), Mϕ, mast cells (MC) and neutrophils play a central role in chronic inflammatory diseases [32,86,87]. Under normal conditions, immature DCs mature and travel to the lymph nodes in response to foreign antigens or other inflammatory signals, where the antigens are presented to T cells. However, the maturation of DCs declines in EM, with it being hypothesized that immunological components form part of the antigen capture and/or presentation activity. This process might be altered in the endometrium of women with EM. Consequently, EM-circulating anti-endometrial antibodies could mask endometrial antigens. As a result, endometrial antigens might not be effectively recognized, with lost endometrial fragments remaining, potentially leading to ectopic establishment [83,88,89].

Mϕ are abundantly recruited to lesions of EM after activation by certain chemokines and cytokines. These cells can release different types of inflammatory substances, creating

an inflammatory microenvironment that contributes to the establishment and growth of endometriotic lesions. In turn, these changes can induce the Mϕ recruitment and as a result, form a vicious circle during the development of the disease [3]. Mϕ are classified as M1 Mϕ, which exhibit proinflammatory activity, and M2 Mϕ, which provide an anti-inflammatory environment and are capable of remodelling tissue through pro-fibrotic activity. In the context of EM, the differentiation between M1 and M2 Mϕ seems to be shifted in favour of M2 in EM and this is particularly important since M2 Mϕ are more immunologically tolerant [20,30,90]. It has been reported that endogenous Mϕ are involved in tissue remodelling during the development of EM, and the M2 Mϕ, in particular, is required for the growth of ectopic lesions in a mouse model [30]. More recently, two independent groups found a decrease in the percentage of M1 and an increase in M2 Mϕ in peritoneal washings of EM patients, especially those with advanced disease (stages III–IV) [91,92].

MCs are known to be key players of the immune system, especially during allergic reactions. However, increasing evidence supports the involvement of these cells also in the inflammatory process of EM. High numbers of degranulated MC have been found in endometriotic lesions showing their influence on EM lesions in development, survival, phenotype, and function via the regulation of other immune cells (monocytes/Mϕ, granulocytes, DC, and T-B lymphocytes) [31–34,93,94].

Neutrophils are considered simple foot soldiers of the innate immune system and are undoubtedly the major effectors of acute inflammation. Several lines of evidence indicate that they also contribute to chronic inflammatory conditions as well as adaptive immune responses [95]. The infiltration of neutrophils into the peritoneal cavity is significantly increased in EM patients compared with that in healthy women, especially in the early stage of EM [96,97]. In EM, neutrophils in the abdominal cavity can secrete an effective pro-angiogenic factor, VEGF, which is also increased in the PF in the EM. As a result, neutrophils may support the growth of endometriotic lesions by secreting VEGF. Moreover, there may be some other nonclassical factors secreted by neutrophils that can promote inflammation and neovascularization in EM [97].

Aberrant expression of several cytokines by inflammatory cells, such as IL-1, IL-4, IL-6, IL-8, IL-10, IL-33, TNFα and growth factors, e.g., transforming growth factor (TGF-β), insulin-like growth factor (IGF-1), hepatocyte growth factor (HGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), have been reported in EM [89,98]. Indeed, cytokines such as IL-8 and TNF-α are known to promote endometrial cell proliferation, endometrial adhesion, and angiogenesis. Furthermore, endometriotic lesions can induce the expression of PGs, MCP1, glycodelin, and other inflammatory mediators [25,99]. Specifically, PGE2, PGF2α, and TNF-α are produced and increased in the early stage; TNF-α, NGF, and IL-17 can cause persistent inflammation; and PGE2, PGF2α, transforming growth factor-β (TGFβ), glycodelin, and TNF-α can induce the sensation of pain [3,25,100]. A recent study showed that cytokine analysis of PFs could differentiate women diagnosed and stratified laparoscopically with ovarian endometrioma, peritoneal, or deep infiltrating EM. This suggests that certain cytokine signatures could be driving different biological signalling events and immune responses in these patients [101]. These inflammation-associated substances act on inflammatory cells in turn. These retroactions lead to more inflammatory cell recruitment in lesions with a subsequent alteration in the original peritoneal and pelvic environments and the formation of a new inflammatory microenvironment. The growth, implantation, infiltration, and migration of EM lesions occur subsequently and retroact on inflammatory cells and substances. This vicious cycle contributes to the aggregation of EM-associated inflammation [3].
