S. F. Ahmad1 , N. Michaud1 , H. Rakhila1 and A. Akoum1
Faculté de Medicine, Endocrinologie de la Reproduction, CHU de Québec, HSFA, Université Laval, Québec, QC, Canada, G1L 3L5
The mechanisms that sustain endometrial tissues at ectopic sites in patients with endometriosis are poorly understood. It is well established now that endometriosis is associated with changes in population and functions of various leukocytes, including macrophages. Macrophages are the most abundant cells found in the peritoneal fluid and are the consistent feature of endometriotic lesion. They infiltrate endometriotic lesions where they undergo alternative activation as a consequence of signals generated within the invaded tissue. However, instead of clearing endometrial cells from the peritoneal cavity and restoring local homeostasis, macrophages appear to enhance their survival and proliferation by secreting growth, remodelling and inflammatory factors which could contribute to the development of endometriosis as well as to the disease-associated chronic pelvic inflammation and symptoms. Thus, unveiling the molecular mechanisms that underlie macrophage dysfunctions is a critical area of research, which would lead to the development of novel medical treatments for endometriosis. In this chapter, we described how macrophages can play a critical role in the pathophysiology of endometriosis not only via their weakened phagocytic functions but also via other major mechanisms revealed to date.
CytokinesEndometriosisIn vivo modelInfertilityMacrophagesPainProstaglandins
Cluster of differentiation
Fibroblast growth factor
Intercellular adhesion molecule-l
((S,R) 3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic methyl ester
Leukocyte function antigen-l
Mitogen-activated protein kinase
Monocyte chemotactic protein-1
Macrophage migration inhibitory factor
Nuclear factor kappa B
Nerve growth factor
Placental growth factor
Regulated on activation, normal T cell expressed and secreted
Steroidogenic acute regulatory protein
Tumour growth factor
Tissue inhibitors of MMPs
Tumour necrosis factor-alpha
Uterine natural killer
Vascular endothelial growth factor
*Author contributed equally with all other contributors.
Endometriosis is associated with changes in the components of the immune system as the peritoneal fluid of women with endometriosis contains increased numbers of immune cells. Dysfunction of the immune system may play an important role in the development of endometriosis since in normal women, menstrual tissue is refluxed into the peritoneal cavity, but it is cleared by immune cells such as macrophages, uterine natural killer (uNK) cells and lymphocytes. However, in women with endometriosis, these subsets of immune cells have been found to be altered in the peritoneal fluid . Macrophages are the most abundant nucleated immune cells found in the peritoneal fluid , and their role in endometriosis has been studied for more than three decades. Haney et al. first reported an increase in the number of peritoneal macrophages in women with endometriosis . Several studies confirmed this observation and further reported an increased activation of these cells in women with endometriosis [4–7]. However, activated peritoneal macrophages in women with endometriosis seem to have a reduced capacity to eliminate misplaced endometrial cells. Paradoxically, they rather seem to facilitate endometrial cell survival, invasiveness and growth by secreting growth, angiogenic and tissue remodelling factors and thereby contribute to the development of endometriosis. Interestingly, it was reported that even peripheral blood monocytes enhance eutopic endometrial cell proliferation in women with endometriosis but suppress endometrial cell proliferation in healthy women . Puzzling at first, this phenomenon is being progressively elucidated. It is thus likely that a combination of defects either in macrophages or in the inherent capability of endometrial cells themselves to resist immune suppression and apoptosis in the peritoneal environment is involved. We also believe that activating positive feedback loops and a mutual crosstalk occur within the pelvic cavity of women with endometriosis (where the disease is frequently found) between local immune cells, particularly macrophages, and misplaced or implanted endometriotic cells, which lead to the establishment of a chronic inflammatory process and a local environment that favours ectopic tissue growth (Fig. 6.1).
Macrophage in endometriosis: proposed model of positive feedback loops
This chapter provides an overview of the current state of knowledge on macrophages in endometriosis, the functional impairment of these key immune cells and their possible role in the pathophysiology of this disease.
6.2 Physiology of Macrophages
Macrophages are mononuclear phagocytic cells derived from blood monocytes . Monocytes originate from haematopoietic stem cells present in bone marrow, which undergo differentiation. They represent immune effector cells, equipped with chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues during infection. Extravasation of monocytes from blood to tissue initiates maturation processes leading to mature macrophages or dendritic cells (DCs) . After migration to peripheral tissue, activated monocytes differentiate to inflammatory DCs and macrophages, which are determined by the inflammatory milieu, and pathogen-associated pattern recognition receptors present on the surface of these immune cells. The developmental origin and the function of tissue macrophage subsets, such as microglia (macrophages in the central nervous system), dermal macrophages and splenic marginal zone and metallophilic macrophages, remain insufficiently understood . Resident macrophages in lymphoid and non-lymphoid tissues are the phagocytic cells involved in steady-state tissue homeostasis, and they undergo local activation in response to various inflammatory and immune stimuli. These macrophages are classified as being “elicited”, as in the antigen-non-specific response to a foreign body or a sterile inflammatory agent or as being “classically activated” or “alternatively activated” by an antigen-specific immune response. It is difficult to distinguish originally resident macrophages from more recently recruited, elicited or activated macrophages, because cells adapt to a particular microenvironment .
6.2.1 Types and Functions
Macrophages are the professional phagocytes expressing numerous membrane adhesion molecules including scavenger receptors such as the cluster of differentiation 36 (CD-36) receptors ensuring targets fixation and endocytosis [9, 13]. Based on membrane-expressed molecules and secretion, macrophages are commonly classified as M1 or M2 phenotype. M1-macrophages are the pro-inflammatory macrophages expressing and secreting cytokines and antimicrobial molecules such as interleukin-1 (IL-1). M2-macrophages are the anti-inflammatory macrophages secreting a majority of growth factors and anti-inflammatory cytokines . It is important to mention that macrophages are dynamic and flexible cells that can change from a phenotype to another depending on their microenvironments . It is a matter of fact that interferon-γ (IFN-γ) and lipopolysaccharide (LPS) generate M1-macrophages, whereas IL-4 and IL-13 activate M2-macrophages . Furthermore, a pallet of phenotype exists between the common M1-macrophages and M2-macrophages activation such as decidual macrophages present in endometrium, which are hybrid M1–M2 phenotype [10, 17, 18]. Scientists finally conclude that the subtle composition of macrophage environment determines their expression profile and thus their role .
6.2.2 Macrophages Along the Menstrual Cycle
Macrophages are ubiquitously present as resident cells monitoring tissue environment and ready to react to any discrete change . Actually, oestrogen (E2) has been discovered as a regulator of M2-phenotype and is responsible for decidual macrophages efficiency especially in pregnancy . Indeed, resident macrophages in endometrium are differentially expressed along the menstrual cycle. During proliferative phase, macrophages represent only 1–2 % of all cells and express tumour growth factor (TGF), IL-10, metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributing to endometrial regeneration, enhancing extracellular matrix turnover and fibroblasts recruitment and proliferation [20, 21]. When progesterone level rises, macrophages represent 3–5 % of cells probably due to resident macrophages’ proliferation. They express numerous chemoattractant molecules such as macrophage chemotactic protein (MCP)-1, regulated on activation, normal T cell expressed and secreted (RANTES) and IL-8 to allow immune cells infiltration into endometrium where they will prepare for the eventual feto-implantation. Finally, progesterone withdrawal, marking menstrual phase, is characterised by an increase in number of macrophages due to chemoattracted monocyte extravasation . These M1-like macrophages secrete MMP-9, tumour necrosis factor-alpha (TNF)-α and many other pro-inflammatory cytokines and proteolytic enzymes, leading to tissue breakdown and menstruation .
In the peritoneal fluid, macrophages are the most abundant cell type. Normally, the peritoneal fluid contains 0.5–20 × 106 leukocytes/ml, of which 85 % are macrophages . In the absence of pathogens, peritoneal fluid macrophages are present at low level and contribute to tissue homeostasis . Their concentration appears to fluctuate during the menstrual cycle and is maximal during the menses . This is most likely related to the menstrual reflux phenomenon, which is common in almost 90 % of reproductive age women during menstruation. Briefly, a small part of endometrial debris travels through fallopian tubes to the peritoneal compartment instead of being evacuated in vaginal secretions [24, 25], where they are normally phagocytised by peritoneal macrophages .
Concisely, macrophages are the phagocytes implicated in an intimate crosstalk between endometrial tissue and the local immune system. Any perturbation in their close environment or intrinsic modifications could trigger profound imbalance in these interactions.
6.3 Pathophysiology of Macrophages in Endometriosis: A Weakened Immune System May Lead to Endometriosis
The discovery that macrophages play a major role in the pathophysiology of endometriosis dates back to 1981 when Dmowski and his group first reported the existence of a deficient cellular immunity in women with endometriosis and a number of functional changes in several immunologic components of the peritoneal fluid . In the same year 1981, other investigators revealed that the number of peritoneal macrophages is increased in infertile women with endometriosis compared with normal women or women with other causes of infertility , and an increased phagocytosis of spermatozoa by peritoneal macrophages in patients with endometriosis was then shown . Later on, it was found that endometriosis is associated with an increased influx of macrophages that undergo further maturation and activation and release active substances such as prostaglandins (PGs), which can have adverse effects on the fertility [5, 28]. Data from other studies confirmed these findings and further suggested that peritoneal macrophages in women with endometriosis are in an advanced level of differentiation, which may interfere with gametes and pre-implantation embryos and contribute to endometriosis-associated subfertility . By the end of 1980s, it was well established that concentration, activation and abnormal maturation of peritoneal macrophages may facilitate the onset of endometriosis. A wide array of factors produced by activated macrophages appear to be involved in endometrial cell survival, adhesion, invasion, proliferation and the formation of endometriotic lesions. A key role for macrophages in decreased immunologic surveillance, recognition and destruction of misplaced endometrial cells and possible facilitation of ectopic endometrial tissue growth seems well plausible.
Many aspects of macrophage involvement in endometriosis-related symptoms have also drawn a considerable interest, but the mechanisms by which activated macrophages cause infertility, menstrual disorders and pelvic pain in women with endometriosis remain unclear.
6.3.1 Suppression of Phagocytosis and Apoptosis by Macrophages
Braun et al. have observed a reduction in the number of macrophages in the eutopic endometrium of women with endometriosis . Their results were surprising because an increase in macrophages’ number in the peritoneal cavity of these women was well documented [6, 29]. Such a phenomenon would reflect altered chemokine gradients that favour macrophage mobilisation to the ectopic environment due to the presence of cyclical, inflammatory stimuli within that environment. Intrinsic resistance to apoptosis, in conjunction with a physiologic disturbance in macrophage trafficking in the eutopic endometrial environment, would be expected to favour the survival of endometrial cells and lead to their establishment in the ectopic sites. Another study has shown a decrease in the capacity of macrophage-mediated cytolysis of misplaced endometrial cells in the peritoneal locations and an increased resistance of these cells to apoptosis .
Taken together, the evidences available to date suggest that defective cytolytic function in macrophages within the peritoneal cavity coupled with the inherent resistance of the endometrial cells to programmed cell death may be fundamental to the pathophysiology of endometriosis.
It is relevant to mention that in women with endometriosis, the ectopic endometrial tissue also seems to escape immune surveillance in the peritoneal cavity. On one hand, several studies have shown that peritoneal macrophages phagocytise and degrade spermatozoids and that peritoneal macrophages isolated from these women have a greater phagocytic ability than those from fertile or infertile women without endometriosis [27, 31]. These results suggest that, if peritoneal macrophages from women with endometriosis enter the reproductive tract via the oviducts, they might adversely influence fertilisation by phagocytising sperm cells . On the other hand, other studies have shown a decreased phagocytic activity against endometrial cells [33, 34]. Thus, instead of eliminating endometrial debris following retrograde menstruation, peritoneal macrophages of patients with endometriosis would favour the development of endometriotic lesions because of the absence of adequate phagocytosis and the large amounts of cytokines and other growth factors released into the peritoneal cavity . It has been suggested that endometrial cells of women with endometriosis contribute to their own survival and escape of immune suppression by secreting various molecules that are capable of interfering with the process of recognition of immune cells . An example of such a phenomenon is the expression of the soluble form of intercellular adhesion molecule (ICAM)-l. ICAM-1 is a co-receptor that interacts with the integrin leukocyte function antigen (LFA)-l present on the surface of leukocytes in order to participate in cell recognition. It has been demonstrated that eutopic endometrial stromal cells of women with endometriosis express a higher concentration of the ICAM-1 soluble form than those of healthy women. In addition, the expression of this form is even higher in ectopic endometrium. Thus, it is possible that the soluble form of ICAM-1 competes with its cellular form to bind to LFA-1, which will disrupt recognition by leukocytes target cells .
In most circumstances, macrophages secrete MMP-9 to destroy the extracellular matrix to disperse endometrial tissues into small pieces . In addition, CD-36 receptor is highly expressed on the cell membrane of macrophages to facilitate phagocytosis of these small fragments of endometrial debris. However, in the presence of high concentration of prostaglandin E2 (PGE2), the expression of MMP-9 and CD-36 is suppressed [38, 39]. This significantly inhibits the phagocytic ability of macrophages and may favour abnormal endometrial tissue growth into the ectopic host sites.
It is worth mentioning here that an apparent inconsistency seems to emerge considering the ability of macrophages to phagocytise sperm cells and their inability to eliminate ectopic endometrial cells. While it is possible that such a difference is at least partly due to inherent properties of endometrial cells of women with endometriosis, the underlying mechanisms remain to be clarified, given their likely impact on the pathogenesis of the disease and the symptoms it causes.
6.3.2 Macrophage Secretions in Endometriosis
Elevated concentrations of cytokines, growth and pro-inflammatory and angiogenic factors have been found in the peritoneal fluid of women with endometriosis. These different factors can originate from a wide variety of sources including endometriotic lesions themselves, macrophages, T cells and follicular fluid after ovulation . IL-1β, IL-8, macrophage migration inhibitory factor (MIF), MCP-1, PGE2 and prostaglandin F2α (PGF2α) are among the main cytokines and eicosanoids that are present at high levels in the peritoneal fluid of women with endometriosis [34, 40–44] and known to be overproduced by activated macrophages. Our previous studies have for the first time detected the existence of endometrial dysfunctions in patients with endometriosis. Thus, endometrial cells are more sensitive to activation by IL-1β, a cytokine that is mainly released by activated monocytes/macrophages and found at high concentrations in the peritoneal fluid of patients with endometriosis . IL-1β, also known as catabolin, can enhance the development of endometriosis by causing the release of factors for blood vessel growth. Other data suggest that IL-1β can also be involved in the up-regulation of MIF expression by ectopic endometrial implants .
IL-8, known as an α-chemokine that shows chemotactic activity and acts as a potent angiogenic agent , is believed to play a significant role in endometrial physiology and endometriosis. Mainly produced by peripheral macrophages as well , IL-8 was detected at higher concentration in the peritoneal fluid of women with endometriosis. In addition to its chemotactic and activating properties for granulocytes, IL-8 was recently found to stimulate proliferation of endometrial cells .
MCP-1 is a P-chemokine having a potent chemotactic activity for monocytes, macrophages and lymphocytes  and produced by different cell types. In endometriosis, however, MCP-1 was found to be over-expressed in eutopic and ectopic endometrial cells as well as in peritoneal fluid macrophages of women with endometriosis [51, 52], and its peritoneal concentrations appeared to correlate with the stage of the disease [53, 54]. This factor may play a role in the development of endometriotic lesions not only by recruiting and stimulating peritoneal macrophages but also by directly stimulating endometrial cell proliferation  and angiogenesis [55, 56]. MCP-1 has potent direct angiogenic effects on endothelial cells  and may act indirectly via its ability to stimulate macrophage recruitment and activation and the release of angiogenic factors [52, 58, 59].
RANTES, one of the members of C–C chemokine family that mediate monocyte chemotactic activity, was found to be elevated in peritoneal fluid of women with endometriosis . RANTES peritoneal fluid levels also correlate with advanced endometriosis stage, suggesting that it might contribute to the progression of this disease . Interestingly, our and other studies showed that macrophage-/monocyte-derived pro-inflammatory cytokines such as IL-1β trigger endometriotic cells to produce this monocyte chemoattractant chemokine [61, 62] and point thereby to feedforward amplification loops underlying chronic pelvic inflammation in women with endometriosis.
MIF, originally identified as a protein factor secreted by T cells, inhibits the migration of macrophages in vitro (hence its name: macrophage migration inhibitory factor) [63, 64]. MIF is actually expressed by most immune cells such as monocytes, macrophages or B cells. It has been shown that MIF plays an important role in the cell-mediated immune response by promoting the Th1 response by the production of IL-12 by macrophages [65, 66]. MIF is able to inhibit the immunosuppressive effects of glucocorticoids on the production of pro-inflammatory cytokines such as IL-1β in activated macrophages [66–69]. To illustrate this property, it has been described that MIF induces the expression of cytoplasmic phospholipase A2 which is an important component of the pro-inflammatory cascade and usually blocked by the action of glucocorticoids . Our studies showed that MIF is overproduced by activated peritoneal macrophages in women with endometriosis  and further found abundant levels of this factor in peritoneal fluid , peripheral blood  and eutopic endometrial tissue of endometriosis patients . Furthermore, MIF levels varied according to endometriosis stage and were particularly elevated in women suffering from endometriosis but also complaining from pelvic pain and/or infertility. This, together with the increased peritoneal fluid levels of MIF, was corroborated by other investigators [72, 73]. Our subsequent studies showed that MIF expression is also elevated in the early stage and highly vascularised endometriotic lesions  and appeared to act at multiple coordinated levels in the PG biosynthesis cascade, thereby inducing cyclooxygenase-2 (Cox-2) expression in these cells and stimulating PGE2 . From a cellular mechanism point of view, our work suggests the involvement of the transcription factor nuclear factor kappa B (NF-κB) in MIF gene activation in ectopic endometrial cells in response to IL-1β . In addition, our group revealed for the first time the presence of a positive feedback loop by which E2 acts directly on ectopic endometrial cells to up-regulate the expression of MIF, which, in turn, displays the capability of inducing the expression of aromatase, the key and rate-limiting enzyme for E2 synthesis . Our studies also revealed that MIF exerts a potent indirect angiogenic effect by interacting with ectopic endometrial cells and inducing the secretion of major angiogenic factors via CD-44 and CD-74 and mitogen-activated protein kinase (MAPK) signalling pathways  and provide evidence for a possible new mechanism underlying endometriosis development and pathophysiology. Angiogenesis or the formation of new blood vessels is essential for the development and the maintenance of endometriotic lesions. Vascular endothelial growth factor (VEGF), one of the major angiogenic factors endowed with the capability of stimulating mitogenesis, migration and differentiation of endothelial cells, is strongly expressed in endometriotic tissue as well as in peritoneal macrophages [78, 79]. Peritoneal-activated macrophages are the major source of VEGF in endometriosis and that this expression is regulated directly by ovarian steroids . Ovarian steroids regulate the production of this growth factor through peritoneal macrophages. E2 acts on various macrophage signalling pathways, influencing in particular those related to sustain the recruitment of inflammatory cells and the remodelling of inflamed tissues, such as MAPK, phosphatidylinositide-3-kinase/protein kinase B and NF-κB. As a consequence, a deregulated response to steroids might influence the survival of ectopic endometrial cells and promote the vascularisation of the lesions [81–85]. It is quite established that hypoxia induces the expression of VEGF [81, 86, 87]. The effects of hypoxia are mainly mediated by hypoxia-inducible factor-1 (HIF-1) protein complex, which is composed of two subunits, HIF-lα, the inducible unit, and HIF-1β, the constitutive unit . In endometriotic tissue, abnormally high levels of complex HIF-lα were found , which makes clear that hypoxia is involved in VEGF production in endometriotic lesions and presumably by peritoneal macrophages.
It is of note that peritoneal macrophages strongly express Cox-2 , one of the rate-limiting enzyme for PGs secretion. As described in several reports [26, 84, 91, 92], PGE2 and PGF2α themselves induce the over-expression of Cox-2 in macrophages and endometriotic stromal cells, leading to an elevated concentration of their own levels in the peritoneal fluid. On one hand, high levels of PGs act on macrophages to suppress their phagocytic ability by down-regulating MMP-9 and CD-36 [37, 39]. On the other hand, PGs stimulate the steroidogenic capacity of endometriotic stromal cells by up-regulating steroidogenic acute regulatory protein (StAR) and aromatase , which induces aberrant biosynthesis of E2. E2 further stimulates the production of mitogens such as fibroblast growth factor-9 (FGF-9), which induces endometriotic cell proliferation [94, 95]. PGE2 can also induce FGF-9 expression by PGE2 receptor 3-dependent transcriptional up-regulation [96, 97]. Other main functions of PGs include the induction of the expression of angiogenic factors such as VEGF and FGF-2 to induce angiogenesis [98, 99]. These effects may play an important role in the survival and proliferation of endometriotic cells. Furthermore, elevated PGs concentrations in endometriosis women is suspected to be involved in pelvic pain and infertility, though more studies will be required to further elucidate the underlying mechanisms [90, 100, 101].
Obviously, macrophages have a rather heterogeneous array of characteristics and dramatically modify their environment not only via the production of cytokines and other pro-inflammatory factors but also via reactive oxygen species (ROS) [102–104]. Production of ROS is known to increase after activation of immune cells, especially macrophages  and their production was reported to be increased in serum and peritoneal fluid of patients with endometriosis. In addition, markers of oxidative stress have been found elevated [102–104]. ROS role, as a second messenger of cellular proliferation, has been described. McCubrey et al. found that normal cell proliferation correlated with production of endogenous ROS through the activation of growth-related signalling pathways . Endometriosis is considered a benign disease but shares some features with cancer, such as propensity to invasion, unrestrained growth, neo-angiogenesis, and distant spreading [19, 107]. The known correlation between ROS and proliferation of cancer cells, along with the increased production of ROS in response to chronic inflammation in endometriosis, thus suggests a possible role for ROS in the regulation of endometriotic cell proliferation.
6.4 Deleterious Effects Induced by Macrophages in Endometriosis
The available literature argues in favour of a significant role for macrophages in the growth and development of endometriotic lesions, the generation of pain through interaction with nerve fibres and infertility via the impediment of spermatozoids and/or oocyte functions and endometrial receptivity. It is obvious that little evidence is available as to the existence of a direct cause and effect relationship between endometriosis-associated macrophage dysfunctions and pain or infertility. However, chronic pelvic inflammation in women with endometriosis and many inflammatory and embryotoxic factors involved, abnormally expressed and known for being secreted by activated endometrial and peritoneal macrophages let, in an indirect way, believe in such a role.
6.4.1 Macrophages in Pain Related to Endometriosis
The pathophysiology of pelvic pain associated with endometriosis and macrophages is unclear, especially that there is no correlation between the existence of pain and the extent of damage. Main inflammatory factors, such as PGs (e.g. PGE2 and PGF2α) or cytokines (e.g. MIF), released by macrophages can also irritate tissues, stimulate nerve fibres and cause severe, painful reaction even when very small areas are involved. The eutopic endometrium of women with endometriosis has higher rates of MIF than the eutopic endometrium of normal women; this is correlated with the degree of advancement of the disease. In addition, this increase of the MIF expression is even more obvious in patients with pelvic pain . It is interesting to note that MIF concentration varies during the menstrual cycle. This variation of expression suggests that MIF would be dependent of ovarian hormones . Recent studies have also shown that MIF stimulates Cox-2 expression and PGE2 production in the ectopic endometrial cells  leading to chronic inflammation.
The density of nerve fibres in peritoneal endometriotic lesions is much higher than in normal peritoneum. Many inflammatory substances can potentially stimulate the nerve endings of these fibres. Recent studies have demonstrated that VEGF can also act as potent neurotrophic factor in addition to its effects on vessels [109, 110]. But the most important growth factor that plays a role in facilitating development, growth and repair of nerve fibre is the nerve growth factor (NGF). An elevated concentration of PGE2 is thought to stimulate production of NGF  which is released from macrophages, as well as endometriotic lesions, and induces smooth muscle metaplasia and innervations. Subsequently, the contraction of smooth muscle cells and hyperalgia of sensory nerve cells, derived from innervations in interstitial lesions, induce endometriotic pain .
PGE2 and PGF2α play an important role in inflammation by increasing the vascular permeability, cause of oedema, by generating a fever or by regulating blood flow and are also involved in the sensation of pain [113, 114]. Over their nociceptive properties, PGs seem to be involved in pelvic pain often present in women with endometriosis. Cyclooxygenase inhibitors, such as nonsteroidal anti-inflammatory drugs, are frequently used in the treatment of mild endometriosis and can significantly reduce pain in these patients . The inhibition of certain macrophage activities may have a benefit in the future relief of endometriosis-related pain .
In conclusion, several relevant studies provide a plausible link between peritoneal inflammation, increased macrophage number and the development of new nerve fibres throughout the peritoneal cavity, which may be associated with the generation of the pelvic pain sensations in women with endometriosis.
6.4.2 Macrophages in Infertility Related to Endometriosis
It is now well known that macrophages are located into decidua (endometrium of the pregnant uterus) during the implantation window and implicated in the physiological processes of implantation, establishment and maintenance of pregnancy and labour control. Indeed, decidual macrophages (20–25 %) and uNK cells are predominant decidual leukocytes [115, 116]. While other uterine leukocytes are diminished during pregnancy, macrophages proportion remains unchanged. During implantation, macrophages cooperate with developing embryo to insure trophoblast endovascular invasion and anchorage. At this site, only M2-macrophages are found infiltrated into endometrial basalis where they will secrete MMP-7 and MMP-9 degrading extracellular matrix to facilitate trophoblast invasion into myometrium . Moreover, M2-macrophages are able to produce VEGF and placental growth factor (PlGF) implicated in the profound remodelling of uterine vasculature during embryonic growth. Furthermore, those phagocytes participate in maternal cell apoptosis and trophoblast renewal. In fact, maternal decidua cell apoptosis insures trophoblast invasion to remodelling and development of embryo. It is important to notice that phagocytosis of apoptotic bodies are vital to avoid secondary necrosis and so a harmful inflammatory response generating tissue damage [118, 119]. Even more, macrophages are fundamental actors of maternal immune tolerance in pregnancy. Indeed, these immune cells are not only able to secrete molecules such as IL-10, known to inhibit T cell activation, but are also to diminish expression of co-stimulatory molecules (CD-80 and CD-86) and indoleamine 2,3-dioxygenase . They create a microenvironment capable of containing immune response to hemi-allogeneic fetal cells even during infection . Interestingly, macrophages secrete IL-15, an uNK cell chemoattractant, and down-regulate uNK cell cytotoxic capacity . uNK cells are crucial for implantation knowing that they are responsible for trophoblast chemoattraction and invasion by secreting IL-8 and IFNγ-inducible protein-10 that binds to trophoblasts’ membrane receptors .
Intriguingly, in endometriosis, the number of M2-macrophages is significantly increased in decidua and peritoneal fluid. As explained in the previous section, macrophages are differentially activated in endometriosis overproducing a myriad of pro-inflammatory factors and key processes are crucial for implantation. Decidual macrophages in endometriosis have been shown to secrete elevated concentrations of angiogenic factors (VEGF, MCP-1, IL-8) and to mediate the development of an anarchic vasculature similar to unstructured tumour vasculature [123, 124]. Such an abnormal angiogenic process might disfavours the functional crosstalk between maternal decidua and trophoblasts, which is essential for embryo survival . Acute and controlled inflammation is necessary for pregnancy, but any chronic inflammation leads to spontaneous abortion . As described previously, women with endometriosis are characterised by an exaggerated inflammation that may contribute to infertility in endometriosis . First, disruption of the phagocytic function in endometriosis may promote accumulation of apoptotic bodies and secondary necrosis responsible for inflammation. Second, the cocktail of inflammatory molecules secreted in endometrium generates an acute inflammation, which is deleterious for trophoblastic implantation. In fact, some relate miscarriage to TNF-α increase and normal pregnancy with high concentrations of IL-10 . Chronic inflammation may inhibit trophoblast invasion and immune tolerance . Macrophage phagocytic function is also an issue since sperm phagocytosis has been described in endometriosis patient. Moreover, an activation of specific sperm engulfment surely prevents or disturbs oocyte fertilisation . It seems that endometrial cells express or secrete molecules able to impair phagocytosis in macrophages, but further investigations could provide interesting insights on this phenomenon. In the meantime, endometriosis is also characterised by an oxidant environment inauspicious for pregnancy . Both endometriotic cells and macrophages are responsible for ROS high concentration in peritoneal fluid of women with endometriosis. More precisely, endometriotic lesions activate inducible nitrogen oxide synthase (iNOS) in macrophages leading to an augmentation of NO production . Moreover, the antioxidant machinery (secreting ascorbic acid, GPx, thiol) is down-regulated creating an imbalance in the redox status of peritoneal environment [132–134]. Interestingly, ROS are known to be related to age-related fertility decline, in vitro fertilisation failure and cigarette smoke subfertility [135–137]. In endometriosis, such redox status may affect sperm viability in peritoneal microenvironment, oocyte and embryo quality .
Since macrophages are crucial cells in implantation and pregnancy, it is not surprising that their dysfunctions may be a significant possible cause of infertility in endometriosis.
6.4.3 Endometriosis, Macrophages and Cancer
In recent years, a possible link between endometriosis and certain types of cancer, such as ovarian cancer, has been suggested. It is well established that endometriosis shares a number of features with cancer such as abnormal cell proliferation and invasion, the development of new blood vessels [138, 139], the decrease in the number of cells undergoing apoptosis  and its stem cell-like activity . Macrophages are important inflammatory mediators and have been implicated in endometriosis through their role in chronic inflammation. As discussed previously in this chapter, macrophages produce a wide range of cytokines which can also induce the progression of tumorigenesis . IL-1 is a potent mediator of inflammation produced by macrophages and it promotes tumorigenesis by stimulating the production of other cytokines and growth factors. Among these, VEGF has been detected in endometriosis-associated ovarian carcinoma . In addition, it has been proposed that VEGF acts as a growth factor for tumours regardless of its role in angiogenesis as there was no correlation observed between VEGF expression and micro-vessel density in ovarian tumours .
Taken together, it seems that macrophages might play a role in the development of cancerous transformation at the site of endometriosis by secreting important mediators which are responsible for tumour progression and development. However, further studies are required to ascertain any possible link between endometriosis and cancer.
6.5 Animal Models to Study the Role of Macrophages in Endometriosis
In order to study endometriosis in vivo, an animal model of the disease is required where endometriosis could be induced experimentally by implanting the human endometrial tissue intra-peritoneally. Since endometriosis occurs spontaneously only in humans and some non-human primates, animal models of induced endometriosis have been developed and are of high value for the evaluation of pathophysiological mechanisms underlying the development of this disease. There are two main groups of animal models for endometriosis: rodents and non-human primates. This section of the chapter will summarise the study of macrophages in the animal models for endometriosis.
Mice and rats are the two established experimental animal models among the rodents. Since rodents do not shed their endometrial tissue and therefore do not develop endometriosis spontaneously, endometriosis can be induced by transplanting endometrial tissue to ectopic sites. These models are classified into two types, homologous and heterologous models. Homologous models have been employed utilising the surgical transplantation of endometrium of the same or syngeneic animals in immunocompetent animals, whereas in heterologous models, human endometrial fragments are transferred either intra-peritoneally or subcutaneously to immunodeficient mice.
Nude and Knockout Mice
As mentioned, animal models represent a useful tool to study in vivo early steps of this disease. The latter approach relies on the transfer of fragments of endometrial tissue harvested from syngeneic donor mice and recapitulates important aspects of the disease . The first experimentally induced endometriosis in mice was reported in 1984 , where normal and ectopic human endometrial tissues were successfully transplanted into the peritoneal cavity of athymic nude mice . This study demonstrated that endometrial tissue keeps intact structure and shows the presence of glands and stroma with an infiltration of macrophages. Since then, several groups have used fluorescent markers such as green fluorescent protein  or bioluminescent markers such as luciferase expression system to generate endometriosis mouse models . Recent studies on Tie-2 knockout mice demonstrated that alternatively activated macrophages can infiltrate endometriotic lesions and promote angiogenesis [146, 147]. These data indicate that the recruited macrophages have more than one effect. Also, it has been shown that knockdown of annexin A2 inhibited the phagocytic function of macrophages, whereas treatment with annexin A2 recombinant protein enhanced phagocytosis . In addition, we have successfully used the athymic nude mice model to study the role of MIF in the development of endometriosis . As described previously in this chapter, MIF has been a regulator of immune system that promotes the pro-inflammatory functions of immune cells, and its role in angiogenesis, tumorigenesis and autoimmune diseases is well established [74, 150–154]. We have reported previously that expression of MIF is increased in eutopic endometrial tissue of women with endometriosis, which is related to the stage of endometriosis . Furthermore, our research in agreement with others has shown a significant elevation in circulating and local peritoneal levels of MIF [43, 44]. Taking the advantage of athymic nude mice model of experimentally induced endometriosis, we have developed a treatment model of endometriosis. In this study, we have used the in vivo model of experimentally induced endometriosis and challenge MIF in order to discover a possible target for the treatment of endometriosis. We used (S,R) 3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic methyl ester (ISO-1), a specific antagonist of MIF  where human endometrial tissue was allowed to implant and grow prior to any treatment. Thus our group is the first to report that macrophage migration inhibitory factor can be a suitable target for the treatment of endometriosis as there is no specific targeted treatment available at present .
Among the rodents, rat is another choice as an experimental model for the endometriosis for the researchers. Matsubavashi et al. reported the first study where rats with auto-transplanted endometrium showed the same immunologic changes as humans with endometriosis . In this study the effect of ectopic endometrial lesions on the changes of leukocyte subpopulation in the rat model of endometriosis was reported. This was supported by another independent research where it was reported that the stromal tissue of uterus-attached peritoneum showed proliferation and infiltration of macrophages in rat endometriosis models . Furthermore, it was discovered that an increase in the number of activated macrophages in the endometriotic lesions has a positive correlation with VEGF . Recently, liposomal bisphosphonate  and a selective Cox-2 inhibitor  have been used as therapeutic drugs targeting macrophages in a rat experimental endometriosis model. These findings have suggested that macrophage depletion effectively inhibits the initiation and growth of endometriotic implants in a rat endometriosis model, and further studies are required to confirm these findings in order to use this approach as a treatment for endometriosis.
6.5.2 Non-human Primates
In the recent past, researchers have started using rhesus macaque (aka rhesus monkey) as an animal model for endometriosis since spontaneous development of the disease requires menstrual shedding. Endometriosis occurs naturally only in some non-human primate species, making development of lesions more comparable to the establishment of disease in humans. Compared with rodents, the non-human primate model of endometriosis is advantageous due to a close recapitulation of human disease and physiology . In a recent study, it has been shown that the activation status of macrophages in endometriosis in the rhesus monkey is more oriented towards the M2 phenotype, in exactly the same way as humans .
Concisely, non-human primates have been extensively used for the investigation of endometriosis, but the very high cost of animal handling limits their use. For this reason, the establishment of rodent models for endometriosis via the intra-peritoneal transplantation of pieces of endometrial tissue has been greatly exploited in the recent years.
6.6 Future Research and Perspectives
Altogether, it is evident that impaired functions of macrophages enable endometriotic tissue proliferation and their secretory products are implicated in the pathophysiology of endometriosis and the major symptoms of the disease such as pain and infertility. Presently, there is an urgent need for new approaches to the medical treatment of endometriosis. Since it is obvious that macrophages are closely associated with endometriosis, future treatment strategies may be based on immunological approaches. Currently, treatments are mostly focused on cytokines and growth factors secreted by macrophages which showed abnormal concentrations in the peritoneal fluid and peripheral blood of endometriosis patients. To regulate angiogenesis, the effect of anti-VEGF antibody has been investigated with an endometriosis mouse model. Bevacizumab, first used for cancer chemotherapy, has been shown to decrease VEGF levels in serum and prevent lesion vascularisation and thus trigger endometriotic cell apoptosis . The most promising molecule is metformin, an anti-inflammatory and aromatase inhibitor known as a biguanide insulin sensitiser. Metformin is able to decrease IL-8, IL-6 and VEGF concentration and improve endometriosis symptoms in women with endometriosis . In the same field, statins seem to prevent MCP-1 expression and to reduce the number and size of endometriotic implants in mice . However, there is no treatment available targeting specifically and directly macrophages. An increased understanding of the immune aspects in endometriosis would be beneficial to set up such novel treatment strategies. Inhibiting MIF might be a promising strategy for future therapies targeting endometriosis, and recent in vivo data from our lab using ISO-1, a potent MIF inhibitor, support this hypothesis . Other approaches to MIF inhibition may include anti-MIF or anti-MIF receptor antibodies. Moreover, macrophages can express and secrete various molecules depending on their activation status. Clearly, in endometriosis, peritoneal macrophages show a different secretory and membrane profile. It would be interesting to target macrophage activation and polarisation in order to control the disease. Actually, some strategies already focus on a modulation of macrophage polarisation. For example, in ovarian cancer administration of INF-γ induces macrophage phenotype modifications promoting tumoricidal activity [165, 166]. Similar treatment strategies applied to endometriosis could open new promising avenues for the management of this disease.
In summary, a growing body of evidence indicates that a combination of endometrial and immune cell dysfunctions plays a major role in the pathogenesis of endometriosis and its major symptoms. Macrophages appear to be critically involved via a wide spectrum of secretory products and functional changes that for the most part remain to be elucidated. A better understanding of the role of macrophages and its intriguing and particular interaction with ectopic endometrial cells is crucial for the development of new medical treatments for this serious disease.
During menstruation, endometrial cells reach the peritoneal cavity as a consequence of retrograde menstruation. These cells resist apoptosis and immune suppression, activate the recruitment of immune cells, particularly macrophages, which have a reduced phagocytic ability, and contribute to angiogenesis, pain innervation, abnormal steroid production and ectopic endometrial cell growth. The reciprocal interactions of misplaced endometrial cells and macrophages create deleterious and activating feedback loops that trigger chronic inflammation and other processes involved in endometriosis pathophysiology.
This study is supported by CIHR grants MOP 93716, 120769 and 123259 to Pr. Ali Akoum, Chercheur National, FRQ-S.
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