**1.1.1 Viable endometrial cells in the peritoneal cavity**

The persistent endometriosis-associated presence of viable peritoneal endometrial cells is partially ascribed to augmented retrograde menstruation, which can directly induce endometriosis, probably by overcoming the immune systems by simple outnumbering (Kyama et al., 2003). Endometrial cell clearance is presumably also reduced by altered phagocytotic properties of peritoneal macrophages. Moreover, decreased cytotoxicity towards autologous endometrial cells by decreased natural killer (NK) cell activity is thought to be an immune defect in endometriosis and is correlated with disease severity (Osuga et al., 2011). The tightly regulated balance between T helper cells type 1 (Th1) and 2 (Th2) is altered towards higher Th2 activity in women with endometriosis. These cells secrete interleukin 4 (IL-4) and IL-10, which are speculated to reduce NK and T-cell cytotoxicity, and thus further reducing immune surveillance.

The endometrial cells themselves also contribute to their own enhanced survival. The transcriptional expression of intercellular adhesion molecule 1 (ICAM-1) is up-regulated in endometriosis lesions, a fact that might contribute to early implantation of peritoneal endometriosis (Wu et al., 2004). The inflammatory cytokines interferon γ (IFNγ) and IL-1β induce ICAM-1 protein expression and the secretion of the soluble form of ICAM-1, which competitively inhibits ICAM-mediated cytotoxicity thus increasing survival. Furthermore, the peritoneal endometrial cells also have a decreased ability to undergo apoptosis.

### **1.1.2 Endometrial-peritoneal adhesion**

The immune system is believed to assist the adhesion of the viable endometrial cells (figure 1). Increased peritoneal infiltration of leukocytes, especially macrophages, and an increased proportion of activated macrophages, is found in endometriosis patients (Haider & Knöfler, 2009). Peritoneal macrophage depletion has been shown to effectively inhibit the initiation and growth of endometriosis implants in rats (Haber et al., 2009), pointing towards a pathogenic effect of macrophages. This inflammation is associated with elevated levels of inflammatory cytokines, growth factors and chemokines.

The pleiotropic pro-inflammatory cytokine tumor necrosis factor α (TNFα) is mainly produced by monocytes and macrophages and stimulates the secretion of the interleukins IL-1, IL-6 and IL-8 by endometriotic cells (Haider & Knöfler, 2009). TNFα can either promote cell regeneration (through the Nuclear Factor kB (NF-κB) pathway) or cell destruction (by initiating the caspase cascade), depending on its local concentration, expression pattern of receptors, and abundance of inhibitors. Macrophages in the peritoneal fluid from women with endometriosis secrete higher levels of TNFα than those from healthy women, and elevated peritoneal fluid levels of TNFα has been detected in women with endometriosis. The adherence of endometrial stromal cells to mesothelial cells is significantly increased by pretreatment of mesothelial cells with TNF-α *in vitro*, suggesting a role in facilitating pelvic adhesion.

peritoneal cavity should be cleared by the immune system, and a number of mechanisms contribute to the failure of this in endometriosis. These mechanisms are summarized in

The persistent endometriosis-associated presence of viable peritoneal endometrial cells is partially ascribed to augmented retrograde menstruation, which can directly induce endometriosis, probably by overcoming the immune systems by simple outnumbering (Kyama et al., 2003). Endometrial cell clearance is presumably also reduced by altered phagocytotic properties of peritoneal macrophages. Moreover, decreased cytotoxicity towards autologous endometrial cells by decreased natural killer (NK) cell activity is thought to be an immune defect in endometriosis and is correlated with disease severity (Osuga et al., 2011). The tightly regulated balance between T helper cells type 1 (Th1) and 2 (Th2) is altered towards higher Th2 activity in women with endometriosis. These cells secrete interleukin 4 (IL-4) and IL-10, which are speculated to reduce NK and T-cell

The endometrial cells themselves also contribute to their own enhanced survival. The transcriptional expression of intercellular adhesion molecule 1 (ICAM-1) is up-regulated in endometriosis lesions, a fact that might contribute to early implantation of peritoneal endometriosis (Wu et al., 2004). The inflammatory cytokines interferon γ (IFNγ) and IL-1β induce ICAM-1 protein expression and the secretion of the soluble form of ICAM-1, which competitively inhibits ICAM-mediated cytotoxicity thus increasing survival. Furthermore,

The immune system is believed to assist the adhesion of the viable endometrial cells (figure 1). Increased peritoneal infiltration of leukocytes, especially macrophages, and an increased proportion of activated macrophages, is found in endometriosis patients (Haider & Knöfler, 2009). Peritoneal macrophage depletion has been shown to effectively inhibit the initiation and growth of endometriosis implants in rats (Haber et al., 2009), pointing towards a pathogenic effect of macrophages. This inflammation is associated with elevated levels of

The pleiotropic pro-inflammatory cytokine tumor necrosis factor α (TNFα) is mainly produced by monocytes and macrophages and stimulates the secretion of the interleukins IL-1, IL-6 and IL-8 by endometriotic cells (Haider & Knöfler, 2009). TNFα can either promote cell regeneration (through the Nuclear Factor kB (NF-κB) pathway) or cell destruction (by initiating the caspase cascade), depending on its local concentration, expression pattern of receptors, and abundance of inhibitors. Macrophages in the peritoneal fluid from women with endometriosis secrete higher levels of TNFα than those from healthy women, and elevated peritoneal fluid levels of TNFα has been detected in women with endometriosis. The adherence of endometrial stromal cells to mesothelial cells is significantly increased by pretreatment of mesothelial cells with TNF-α *in vitro*, suggesting a role in facilitating pelvic

the peritoneal endometrial cells also have a decreased ability to undergo apoptosis.

figure 1 and will be addressed here in the order of disease progression.

**1.1.1 Viable endometrial cells in the peritoneal cavity** 

cytotoxicity, and thus further reducing immune surveillance.

inflammatory cytokines, growth factors and chemokines.

**1.1.2 Endometrial-peritoneal adhesion** 

adhesion.

Fig. 1. The role of the immune system in the development and maintenance of endometriosis. Following retrograde menstruation, multiple altered actions of the immune system influence step-wise disease progression. PF, peritoneal fluid; NK cells, natural killer cells; ICAM, Intercellular Adhesion Molecule; TNF, Tumour Necrosis Factor; IL, interleukin; MCP, Monocyte Chemotactic protein; RANTES, Regulated upon Activation, Normal T-cell Expressed and Secreted; TGF, Transforming Growth Factor; MMP, Matrix Metalloproteinase; TIMPs, Tissue Inhibitor of Matrix Metalloproteinases; VEGF, Vascular Endothelial Growth Factor; DC, dendritic cells. Simplified overview inspired by (Kyama et al., 2003).

TNFα also stimulates the growth of both endometrial and endometriotic stromal cells from women with endometriosis, whereas it inhibits the growth of endometrial cells from healthy women (Haider & Knöfler, 2009). The peritoneal fluid level of TNFα is correlated with disease severity. In addition, anti-TNFα therapy effectively reduces endometriosis lesions in rats and baboons (Zulfikaroglu et al., 2010) revealing a potential future medical treatment for endometriosis. However, a placebo-controlled clinical trial of anti-TNFα treatment failed to demonstrate a significant decrease of pain associated with deep endometriosis (see Haider & Knöfler, 2009).

Virus Infection and Type I Interferon in Endometriosis 249

TGF-β might not only facilitate adhesion but also implantation in endometriosis (figure 1). Its expression in the endometrium varies with the menstrual cycle, peaking at the time of menstruation (Omwandho et al., 2009). This expression pattern is roughly opposite to that of TNFα, and co-expression with progesterone occurs in the secretory phase (figure 2). TGFβ has been shown to mediate progesterone-associated suppression of matrix metalloproteinase (MMP) expression in endometrial tissue, and it is required for progesterone action in the prevention of experimental endometriosis (Bondza et al., 2009).

Fig. 2. Levels of estrogen, progesterone, TGFβ, TNFα, and IFNα in the human endometrium during menstrual cycling. Numbers denote days after the first menstrual day. Dashed lines show levels following implantation. There are indications that TNFα and IFNα levels are up-regulated in early pregnancy. TGF, Transforming Growth Factor; TNF, Tumour Necrosis Factor; IFN, interferon. Schematic representation based on (Li et al., 2001; Haider & Knöfler,

**1.1.3 Ectopic implantation and invasion** 

2009; Omwandho et al., 2009).

IL-1β and IL-6 are also a pro-inflammatory cytokine found elevated in the peritoneal fluid of women with endometriosis, and macrophages in the peritoneal fluid produce IL-1β *in vitro* in the absence of stimulants (Bondza et al., 2009). Both IL-1α and IL-1β as well as IL-6 are involved in the activation of T-lymphocytes and the differentiation of B-lymphocytes, thereby contributing to the increased peritoneal active immune state in endometriosis.

In addition to these cytokines, a number of chemokines have been implicated in the pathogenesis of endometriosis, believed to contribute by recruiting and activating leukocytes to the inflammatory sites, thereby assisting the adhesion process. Yet another member of the interleukin family, namely the chemokine IL-8 (also named CXCL-8) has been implicated in endometriosis (Bondza et al., 2009). Macrophages in the peritoneal fluid from women with endometriosis secrete higher levels of IL-8 than those from healthy women, which stimulate the growth of endometrial and endometriotic stromal cells. Elevated levels of IL-8 were found in the peritoneal fluid of women with endometriosis, and concentrations correlated with the severity of the disease. IL-8 stimulates the adhesion of endometrial stromal cells to fibronectin (see Kyama et al., 2003) and is therefore likely to contribute to the adhesion of endometrial cells in the pelvic cavity.

Monocyte chemotactic protein-1 (MCP-1, also named CCL2) is one of the most potent chemokines in attracting monocytes. MCP-1 has been found elevated in both peritoneal fluid and serum from women with endometriosis, and in the endometrial glands the level varies according to disease stage (Bondza et al., 2009). Moreover, estrogen up-regulates IL-1 induced MCP-1 expression, which may occur locally in the inflammatory site and contribute to peritoneal macrophage recruitment and activation.

The chemokine RANTES (regulated upon activation, normal T cell expressed and secreted, also named CCL5) plays an important role in recruiting several types of lymphocytes into the endometrium (Fang et al., 2009). It is secreted by stromal cells at the endometriosis lesions upon stimulation with TNFα, IFNγ, or IL-1β. IL-1β treatment yields a higher level of RANTES in endometriotic compared with endometrial cells from endometriosis patients, which in turn display higher levels than normal endometrial cells, entailing increased monocyte chemotactic activity. This is presumed to be a major contributor to leukocyte recruitment and inflammation in the pathogenesis of endometriosis.

It should be noted that the results on whether cytokines and chemokines are involved are equivocal. However, accumulating evidence predominantly reports that both cytokines and chemokines through enhanced local inflammation and cellular adhesion contribute to the pathogenesis of endometriosis.

Transforming growth factor β (TGFβ) peritoneal fluid levels are significantly higher in women with endometriosis compared with healthy control women, and the TGFβ level increases with the severity of the disease (Liu et al., 2009). The adhesion of human endometrial cells to murine peritoneum is increased by treatment with TGFβ (as well as with IL-1β, IL-6, and TNFα) (Omwandho et al., 2009). The same could not be shown for human peritoneal mesothelial cells, but TGFβ enhances trans-mesothelial invasion by primary and immortalized endometrial epithelial cell lines *in vitro* (Liu et al., 2009).

#### **1.1.3 Ectopic implantation and invasion**

248 Endometriosis - Basic Concepts and Current Research Trends

IL-1β and IL-6 are also a pro-inflammatory cytokine found elevated in the peritoneal fluid of women with endometriosis, and macrophages in the peritoneal fluid produce IL-1β *in vitro* in the absence of stimulants (Bondza et al., 2009). Both IL-1α and IL-1β as well as IL-6 are involved in the activation of T-lymphocytes and the differentiation of B-lymphocytes, thereby contributing to the increased peritoneal active immune state in endometriosis.

In addition to these cytokines, a number of chemokines have been implicated in the pathogenesis of endometriosis, believed to contribute by recruiting and activating leukocytes to the inflammatory sites, thereby assisting the adhesion process. Yet another member of the interleukin family, namely the chemokine IL-8 (also named CXCL-8) has been implicated in endometriosis (Bondza et al., 2009). Macrophages in the peritoneal fluid from women with endometriosis secrete higher levels of IL-8 than those from healthy women, which stimulate the growth of endometrial and endometriotic stromal cells. Elevated levels of IL-8 were found in the peritoneal fluid of women with endometriosis, and concentrations correlated with the severity of the disease. IL-8 stimulates the adhesion of endometrial stromal cells to fibronectin (see Kyama et al., 2003) and is therefore likely to contribute to the adhesion of endometrial cells in the pelvic

Monocyte chemotactic protein-1 (MCP-1, also named CCL2) is one of the most potent chemokines in attracting monocytes. MCP-1 has been found elevated in both peritoneal fluid and serum from women with endometriosis, and in the endometrial glands the level varies according to disease stage (Bondza et al., 2009). Moreover, estrogen up-regulates IL-1 induced MCP-1 expression, which may occur locally in the inflammatory site and contribute

The chemokine RANTES (regulated upon activation, normal T cell expressed and secreted, also named CCL5) plays an important role in recruiting several types of lymphocytes into the endometrium (Fang et al., 2009). It is secreted by stromal cells at the endometriosis lesions upon stimulation with TNFα, IFNγ, or IL-1β. IL-1β treatment yields a higher level of RANTES in endometriotic compared with endometrial cells from endometriosis patients, which in turn display higher levels than normal endometrial cells, entailing increased monocyte chemotactic activity. This is presumed to be a major contributor to leukocyte

It should be noted that the results on whether cytokines and chemokines are involved are equivocal. However, accumulating evidence predominantly reports that both cytokines and chemokines through enhanced local inflammation and cellular adhesion contribute to the

Transforming growth factor β (TGFβ) peritoneal fluid levels are significantly higher in women with endometriosis compared with healthy control women, and the TGFβ level increases with the severity of the disease (Liu et al., 2009). The adhesion of human endometrial cells to murine peritoneum is increased by treatment with TGFβ (as well as with IL-1β, IL-6, and TNFα) (Omwandho et al., 2009). The same could not be shown for human peritoneal mesothelial cells, but TGFβ enhances trans-mesothelial invasion by

primary and immortalized endometrial epithelial cell lines *in vitro* (Liu et al., 2009).

to peritoneal macrophage recruitment and activation.

pathogenesis of endometriosis.

recruitment and inflammation in the pathogenesis of endometriosis.

cavity.

TGF-β might not only facilitate adhesion but also implantation in endometriosis (figure 1). Its expression in the endometrium varies with the menstrual cycle, peaking at the time of menstruation (Omwandho et al., 2009). This expression pattern is roughly opposite to that of TNFα, and co-expression with progesterone occurs in the secretory phase (figure 2). TGFβ has been shown to mediate progesterone-associated suppression of matrix metalloproteinase (MMP) expression in endometrial tissue, and it is required for progesterone action in the prevention of experimental endometriosis (Bondza et al., 2009).

Fig. 2. Levels of estrogen, progesterone, TGFβ, TNFα, and IFNα in the human endometrium during menstrual cycling. Numbers denote days after the first menstrual day. Dashed lines show levels following implantation. There are indications that TNFα and IFNα levels are up-regulated in early pregnancy. TGF, Transforming Growth Factor; TNF, Tumour Necrosis Factor; IFN, interferon. Schematic representation based on (Li et al., 2001; Haider & Knöfler, 2009; Omwandho et al., 2009).

Virus Infection and Type I Interferon in Endometriosis 251

Only a few studies have addressed the possible involvement of a pathogenic virus in the aetiology of endometriosis (Oppelt et al., 2010; Vestergaard et al., 2010). The presence of specific human herpes viruses, human polyomaviruses, and human papillomaviruses was analysed. Also, the presence of human endogenous retroviruses have been investigated in endometriosis lesions (Hu et al., 2006; Oppelt et al., 2009), and these studies will be

The *Herpesviridae* are a large, diverse family of double-stranded, enveloped viruses. Eight human herpesviruses (HHVs) have been identified: Epstein-Barr virus (EBV, of the subfamily *Gammaherpesvirinae*), cytomegalovirus (CMV or HHV5 of the subfamily *Betaherpesvirinae*), herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 of the subfamily *Alphaherpesvirinae*), varicella zoster (VZV), HHV6, HHV7, and the Karposi's sarcoma herpes virus (KHSV or HHV8). The seroprevalence of each of these herpes viruses varies according to a number of demographic factors (Prober, 2011). For EBV, CMV, and HSV-1 the prevalences range from 50 to 75%, whereas the prevalence for HSV-2 is about 25%. For VZV, HHV6 and HHV7 the seroprevalence is almost 100%, whereas the prevalence for HHV8 is less than 10%. The *Herpesviridae* family are found in many human tissue types, including the fallopian tubes (salpingitis) and the endometrium (see Vestergaard et al., 2010). These viruses are commonly distributed as asymptomatic infectious agents but are also all associated with diseases, such as genital sores or a variety of malignancies. EBV can cause infectious mononucleosis and has been associated with lymphomas of B, T and NK cell origin, but also cancers of epithelial origin like nasopharyngeal carcinomas and gastric adenocarcinomas (Dolcetti & Masucci, 2003). EBV maintains latency in B cells. CMV can cause pneumonitis and delayed neurological complications like sensory neural hearing deficits and learning disabilities (Brown & Abernathy, 1998). CMV maintains latency in monocytes and macrophages. HSV-1 and HSV-2 can cause encephalitis and genital ulcerative disease (Wilson et al., 2009). The HSVs maintain latency in neuronal cells. VSV cause chickenpox (varicella) as primary infection following which it becomes latent in neuronal cells (Kennedy & Cohrs, 2010). VZV can be reactivated and thus cause herpes zoster (shingles) and extremely painful vesicular eruption. HHV6 cause roseola during the primary infection (Prober, 2011). HHV6 becomes latent in salivary glands, the brain and in mononuclear cells or macrophages. Most reactived HHV6 infections are

The presence of most of the herpes viruses in endometriosis has been analysed., Endometriosis samples from 32 patients were analysed for the presence of EBV, CMV, HSV-1 and HSV-2 by multiplex PCRs (Vestergaard et al., 2010). None of the clinical samples were positive for any of these four herpes viruses. In another study, 66 endometriosis samples from 56 patients were tested for the presence of EBV, CMV, HSV-1, HSV-2, VZV, and HHV6 by a PCR based analysis (Oppelt et al., 2010). Also this study failed to detect the presence of any of these six herpes viruses. Apart from HHV7 and HHV8, which has not yet been analysed, the six herpes viruses EBV, CMV, HSV-1, HSV-2, VZV, and HHV6 do not seem to

discussed in the following section.

**2.1 Herpes viruses** 

asymptomatic.

be involved in the pathology of endometriosis.

MMPs are important for the control of extracellular matrix turnover, and they are believed to influence the implantation of adhered endometriotic cells in endometriosis (Kyama et al., 2003). The level of MMPs is in up-regulated and tissue inhibitors of matrix metalloproteinases (TIMPs) down-regulated in the peritoneal fluid from women with endometriosis compared with controls (Bondza et al., 2009). MMPs are upregulated in response to the inflammatory cytokines TNFα and IL-1 and are expressed during the proliferative and menstrual phases but suppressed by progesterone in the secretory phase. In the absence of a normal progesterone response of the endometriosis lesions, sensitivity to TGFβ may be altered, and this may result in a failure to down-regulate MMPs, thus contributing to the implantation failure.

### **1.1.4 Growth and maintenance of endometriosis**

Following implantation, the growth and maintenance of endometriosis lesions is influenced by a number of factors (figure 1). First of all, the implanted tissue is in need of vascularization and an increase in the concentration of several angiogenic factors has been reported. IL-6 and IL-8, which as stated above are up-regulated in endometriosis, both promote angiogenesis (Bondza et al., 2009). IL-1β, which is also elevated in the peritoneal fluid of endometriosis patients, induces IL-6, the vascular endothelial growth factor (VEGF), and an angiogenic phenotype in endometriotic but not endometrial stromal cells. Women with moderate and severe endometriosis have elevated levels of both IL-6 and VEGF in the peritoneal fluid compared with healthy controls (Kyama et al., 2003), underlining that angiogenesis is a feature of disease exacerbation. Furthermore, high proliferative activity of endometriosis lesions is associated with increased levels of VEGF and its receptor as well as increased microvessel density (Bondza et al., 2009).

A positive feedback of activated immune response contributes further to the maintenance of endometriosis. The reduced NK cell activity has been suggested to result in less effective killing of autologous dendritic cells (DCs) loaded with endometrial self-antigens. This would facilitate self-antigen presentation to autoreactive T cells and the subsequent production of autoantibodies (Kyama et al., 2003), pointing towards a pathogenic contribution by an autoimmune disease mechanism. Indeed, the frequency of autoantibodies towards endometrial antigens is elevated in both serum and peritoneal fluid of women with endometriosis compared with healthy women (Osuga et al., 2011). Activation of Th2 immune response in endometriosis and elevated B cell levels has been reported both systemically and locally in the endometriosis lesions, supposedly causing sustained autoantibody production and continuous infiltration of immune cells, thus maintaining the immunological contribution to the pathogenesis of endometriosis.
